Antibodies to oxidation-specific epitopes

ABSTRACT

The disclosure provides for single chain variable fragments to MAA-oxidized specific epitopes (OSEs). The disclosure also provides single chain virable fragments that bind to MDA-OSEs or MAA-OSEs on oxidized phospholipids and methods of use thereof, including the production of transgenic animal models and the use of the fragments as therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 from ProvisionalApplication Ser. No. 62/384,694, filed Sep. 7, 2016, the disclosures ofwhich are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Grant Nos.HL119828, HL56989 and HL088093 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Accompanying this filing is a Sequence Listing entitled“Sequence_ST25.txt”, created on Sep. 7, 2017 and having 40.4 kB of data,machine formatted on IBM-PC, MS-Windows operating system. The sequencelisting is hereby incorporated by reference in its entirety for allpurposes.

TECHNICAL FIELD

This invention provides monoclonal antibodies directed tooxidation-specific epitopes, specifically malondialdehyde-acetaldehyde(MAA) that is involved in a variety of diseases, includingcardiovascular disease, liver disease and neurological diseases. Thedisclosure also provides methods of using the antibody as a molecularimaging agent and therapeutic (“biotheranostic”) in these diseasestates. The invention also provides a method of “passive vaccination” toprevent diseases, i.e. atherosclerosis or liver disease.

BACKGROUND

Atherosclerosis is the underlying cause of cardiovascular diseases. Themain pathways that drive this disease are the accumulation of lipids andconcomitant immune cell infiltration in the vessel wall. Oxidizedlow-density lipoprotein (oxLDL) plays a major role in initiation andprogression of so-called atherosclerotic plaques. To detect(sub)clinical atherosclerosis, a variety of imaging techniques areavailable. However, current clinical techniques do not characterize theatherosclerotic plaque, but merely predict the risk of future eventsbased on the level of stenosis. In the past decade, molecular imaginghas provided new insights in pathophysiology at a cellular and molecularlevel. It therefore allows phenotyping and identifying features ofplaque progression that precede a possible rupture.

SUMMARY

The disclosure provides an isolated antibody or antibody fragment thatrecognizes and binds to an MAA oxidized specific epitope (OSE)(MAA-OSE),wherein the antibody or antibody fragment comprises a variable heavychain (V_(H)) domain and/or a variable light chain (V_(L)) domain, andwherein, (a) the V_(H) domain comprises a sequence selected from thegroup consisting of (i) at least one CDR selected from the groupconsisting of SEQ ID NO:25, 26 and 27; (ii) at least one CDR selectedfrom the group consisting of 28, 29 and 30; (iii) at least one CDRselected from the group consisting of SEQ ID NO:31, 32 and 33; (iv) atleast one CDR selected from the group consisting of SEQ ID NO:34, 35 and36; and (v) at least one CDR selected from the group consisting of SEQID NO:37, 38 and 39; and/or (b) the V_(L) domain comprises a sequenceselected from the group consisting of: (i) at least one CDR selectedfrom the group consisting of SEQ ID NO:40, 41 and 42; (ii) at least oneCDR selected from the group consisting of SEQ ID NO:43, 44 and 45; (iii)at least one CDR selected from the group consisting of SEQ ID NO:46, 47and 48; (iv) at least one CDR selected from the group consisting of SEQID NO:49, 50 and 52; and (v) at least one CDR selected from the groupconsisting of SEQ ID NO:52, 53 and 54. In one embodiment, the antibodyor antibody fragment comprises a variable heavy chain sequence of (a)selected from the group consisting of (1) SEQ ID NO:4; (2) SEQ ID NO:8;(3) SEQ ID NO:12; (4) SEQ ID NO:16; and (5) SEQ ID NO:20. In another orfurther embodiment, the antibody comprises a variable light chainsequence of (b) selected from the group consisting of: (6) SEQ ID NO:2;(7) SEQ ID NO:6; (8) SEQ ID NO:10; (9) SEQ ID NO:14; and (10) SEQ IDNO:18. In another embodiment, the antibody comprise a variable lightchain sequence that is at least 98% identical to SEQ ID NO:2 and avariable heavy chain sequence that is at least 98% identical to SEQ IDNO:4. In yet another embodiment, the antibody comprise a variable lightchain sequence that is at least 98% identical to SEQ ID NO:6 and avariable heavy chain sequence that is at least 98% identical to SEQ IDNO:8. In still another embodiment, the antibody comprise a variablelight chain sequence that is at least 98% identical to SEQ ID NO:14 anda variable heavy chain that is at least 98% identical to SEQ ID NO:16.In another embodiment, the antibody comprise a variable light chain thatis at least 98% identical to SEQ ID NO:18 and a variable heavy chainthat is at least 98% identical to SEQ ID NO:20. In yet anotherembodiment, the antibody is non-human and comprises a variable lightchain that is at least 98% identical to SEQ ID NO:10 and a variableheavy chain that is at least 98% identical to SEQ ID NO:12. In a furtherembodiment, the antibody is humanized. In still another embodiment ofany of the foregoing, the heavy and light chain domains are linked to anFc region.

The disclosure also provides a single chain variable fragment (“scFv”)that recognizes an MAA-OSE and comprises (A) a V_(H) domain containing asequence selected from the group consisting of (i) at least one CDRselected from the group consisting of SEQ ID NO:25, 26 and 27; (ii) atleast one CDR selected from the group consisting of 28, 29 and 30; (iii)at least one CDR selected from the group consisting of SEQ ID NO:31, 32and 33; (iv) at least one CDR selected from the group consisting of SEQID NO:34, 35 and 36; and (v) at least one CDR selected from the groupconsisting of SEQ ID NO:37, 38 and 39; and (B) a V_(L) domain containinga sequence selected from the group consisting of (i) at least one CDRselected from the group consisting of SEQ ID NO:40, 41 and 42; (ii) atleast one CDR selected from the group consisting of SEQ ID NO:43, 44 and45; (iii) at least one CDR selected from the group consisting of SEQ IDNO:46, 47 and 48; (iv) at least one CDR selected from the groupconsisting of SEQ ID NO:49, 50 and 52; and (v) at least one CDR selectedfrom the group consisting of SEQ ID NO:52, 53 and 54. In one embodiment,the scFv is soluble under physiological conditions.

The disclosure also provides an antibody comprising a variable lightchain and variable heavy chain sequence selected from the groupconsisting of (a) SEQ ID NO:2 and 4; (b) SEQ ID NO:6 and 8; (c) SEQ IDNO:10 and 12; (d) SEQ ID NO:14 and 16; and (e) SEQ ID NO:18 and 20. Inone embodiment, the variable light chain comprises SEQ ID NO:10 and thevariable heavy chain comprise SEQ ID No:12 and an Fc region is human orhumanized. In another embodiment, the variable light chain and variableheavy chain are human or humanized and the Fc region is non-human.

The disclosure also provides a polynucleotide that encodes an antibody,antibody fragment, variable light chain, variable heavy chain or scFv asdescribed herein.

The disclosure also provides a vector comprising a polynucleotidesequence selected from the group consisting of (a) SEQ ID NO:1 and/or 3;(b) SEQ ID NO:5 and/or 7; (c) SEQ ID NO:9 and/or 11; (d) SEQ ID NO:13and/or 15; and (e) SEQ ID NO:17 and/or 19.

The disclosure also provides host cells transfected or transformed witha vector or polynucleotide of the disclosure.

The disclosure also provide a transgenic animal, comprising apolynucleotide of the disclosure wherein the non-human transgenicorganism expresses an antibody or antibody fragment of the disclosure.

The disclosure also provides a method of treating a subject with anMAA-related disease or disorder comprising administering an antibody orantibody fragment as described herein to the subject, wherein theantibody or antibody fragment binds to and inhibits the biologicaleffect caused by an MAA adduct. In one embodiment, the antibody orantibody fragment comprises a sequence selected from the groupconsisting of: (a) SEQ ID NO:2 and 4; (b) SEQ ID NO:6 and 8; (c) SEQ IDNO:14 and 16; and (d) SEQ ID NO:18 and 20. In one embodiment, thesubject is a human. In another embodiment, the subject has acardiovascular disease or disorder, a fatty liver disease or disorder,an acute lung injury disease or disorder, or rheumatoid arthritisdisease or disorder.

The disclosure also provides a method of treating oxidative stress in asubject comprising administering an antibody or antibody fragment of thedisclosure to the subject, wherein the antibody binds to and inhibitsthe biological effect caused by an MAA adduct.

The disclosure also provides a method of diagnosing an inflammatoryfatty liver disease or disorder comprising contacting a subject with anantibody or antibody fragment of as described herein, wherein theantibody is labeled with a detectable label and imaging the subject todetermine the amount or presence of the antibody in the liver of thesubject. In one embodiment, if the amount of the antibody in the liverexceeds a normal control amount then the subject has an inflammatoryliver disease. In another embodiment, the inflammatory liver disease isnon-alcoholic fatty liver disease. In a further embodiment, theinflammatory liver disease is non-alcoholic steatohepatitis (NASH).

The disclosure also provides a method of diagnosing a MAA-relateddisease or disorder in a subject comprising obtaining a sample from thesubject and contacting the sample with an antibody or antibody fragmentas described herein and measuring the amount of antibody or antibodyfragments bound to and MAA adduct in the sample compared to a normalcontrol sample, wherein an amount of antibody or antibody fragment boundto an MAA adduct in the sample is greater than the control, the subjecthas an MAA-related disease or disorder.

The disclosure also provides a method of treating or inhibitingatherogenesis in a subject, the method comprising administering to thesubject an antibody or antibody fragment of the disclosure, wherein theantibody binds to an inhibits that uptake and/or inflammatory responsecaused by an MAA adduct. In one embodiment, the MAA adduct is associatedwith a molecule in an atherosclerotic plaque.

The disclosure also provides a method for treating atherosclerosisand/or fatty liver disease in a subject, the method comprisingadministering to the subject antibodies or antibody fragments asdescribed herein that specifically bind an MAA adduct, wherein theantibodies or antibody fragments are in a pharmaceutically acceptablecarrier.

The disclosure also provides a method of inhibiting the progression ofnon-alcoholic fatty liver disease to non-alcoholic steatoheptatis (NASH)comprising administering to a subject in need of such treatment andantibody or antibody fragment of the disclosure in an amount to reduceinflammation and to improve biomarkers of liver function.

DESCRIPTION OF DRAWINGS

FIG. 1A-D shows pictorial depiction of MAA-LDL (A). The configuration ofsoluble LA25 Fab antibody fragment. The LA25 lambda light-chain andheavy-chain with a hexa-histidine and the influenza hemagglutinin (HA)epitope tag for detection and purification were expressed under thedirection of lacZ promoter for phage display or Fab production in E.coli. Proteolysis of the ompA and pelB signal peptides in the periplasmgenerated the native amino terminus of Fab and facilitated the joiningof heavy- and light-chains together by disulfide bonds as bio-activesoluble Fab (B). Binding of LA25 to a variety of oxidation-specificepitopes, while LA24 does not (C). Competition assays for thespecificity of LA25 binding to MAA-LDL. LA25 was incubated in theabsence and presence of increasing amounts of indicated competitors, andthe extent of binding to plated MAA was determined. Data are expressedas a ratio of binding in the presence of competitor (B) divided byabsence of competitor (B₀) (D).

FIGS. 2-3 shows sequences (nucleic acid and polypeptide) for thevariable light and heavy domains of an antibodies of the disclosure.FIG. 2 provides SEQ ID Nos:1-4, while FIG. 3 provides SEQ ID Nos: 55-56.

FIG. 4 presents the staining of atherosclerotic lesions from rabbitaorta with selected Fab clones (2^(nd) antibody: anti-HA-biotinconjugate clone HA-7).

FIG. 5 shows MAA specificity of LA25 antibody.

FIG. 6 shows the binding of LA25, KA2 and ML7 Fab to apoptotic cells,which was also confirmed by ELISA. Other panels show, e.g., LA25 doesnot bind normal cells, and that LA24 does not bind normal or apoptoticcells.

FIG. 7 presents deconvolution microscopy of LA25 binding to apoptoticJurkat cells but not normal cells. (Blue—nuclei stained with Hoechstdye; Green—FITC-labeled anti-Fab).

FIG. 8 displays staining of atherosclerotic lesions from WHHL rabbitsand high fat cholesterol diets fed LDLR−/− mice with LA25 and LA24control Fab.

FIG. 9 provides a schematic illustration of MDA-lysine adducts.

FIG. 10 presents competition by oxidation-specific antigens to thebinding of pooled umbilical cord plasma IgM (n=7) to MDA-LDL, MAA-LDL,and MAA-BSA respectively.

FIG. 11 demonstrates that complex MDA-derived epitopes are a majortarget for IgM in human newborn plasma. As shown, preabsorption of IgMin human umbilical cord plasma with oxidation-specific antigens showsthat the MAA-derived adducts constitute a dominant target for naturalantibodies (Nabs) in newborn babies. Data shown as mean±SEM (n=7plasmas).

FIG. 12 demonstrates that human Fabs bind to differentoxidation-specific epitopes in OxLDL. Antigen-profiling and competitionELISA revealed that LA24 is not hapten-specific in contrast to the otherFabs. LA25 and ML7 recognize a MAA-lysine epitope. KA2 and MK17 sharebinding specificity, but to different, though related, epitopes presentin various OxLDL antigens.

FIG. 13 demonstrates that hapten-specific Fabs (LA25, KA2, ML7, andMK17) bind to apoptotic cells and stain rabbit atherosclerotic lesionsand inflamed human rheumatoid synovial tissue, suggesting the presenceof ‘complex’ MDA-derived epitopes. As shown, Fab binding to apoptoticmurine thymocytes by FACS.

FIG. 14 presents immunostaining of frozen rabbit aorta. Human Fabs werevisualized using biotinylated F(ab′)₂ fragments of anti-huIgG[F(ab′)₂-specific], ABC-AP VectaStain, and Vector RED substrate. Nucleiwere counterstained using Hematoxylin.

FIG. 15 presents immunostaining of oxidation-specific epitopes in humaninflamed rheumatoid synovial tissue with Fabs of the disclosure. Thehuman Fabs were visualized as described in FIG. 14, except ananti-HA-tag 2^(nd) Ab was used.

FIG. 16 demonstrates that the Fabs are specific for ‘complex’MDA-derived epitopes compete for modified LDL binding to scavengerreceptors of macrophages. As shown, Fab competition of biotinylatedOxLDL ligand binding to J774 macrophages were assessed in a microtiterplate-based assay.

FIG. 17 demonstrates that LA25 inhibits in vivo-phagocytosis ofapoptotic cells by macrophages. As shown, LA25, but not LA24, inhibitsin vivo-phagocytosis of apoptotic murine thymocytes by elicitedperitoneal macrophages in Rag1^(−/−)xLdlr^(−/−) mice.

FIG. 18 demonstrates the in vivo aortic uptake of radiolabeled LA25. Asshown, an en face preparation of a Sudan IV-stained aorta (bottom) andcorresponding autoradiograph (top) from a male ApoE^(−/−) mouse aftertail vein injection of 10 mCi ¹²⁵I-LA25.

FIG. 19 demonstrates that antibodies of the disclosure bind to uniqueMAA-epitope. Various biotin modified peptides with different number oflysine residues defined by x were modified with MAA and bound toavidin-coated wells.

FIG. 20 shows immunostaining of an early fibroatheroma, a thick capfibroatheroma with multiple rupture and healing and a ruptured plaquewith LA25. The top row displays an early fibroatheroma, the middle row acomplex thick cap fibroatheroma and bottom row a ruptured thin capfibroatheroma stained with H&E, Movat pentachrome, LA25 and LA24antibody control. Note the presence of thrombus in this section (arrow),which also stains with LA25.

FIG. 21 shows immunostaining of LA25 in debris from distal protectiondevices. The bottom row displays serial sections of embolized plaquedebris captured with a distal protection device following percutaneouscoronary intervention of a stenotic 12-year old coronary saphenous veingraft in a patient with crescendo angina. The material is stained withLA25 for MAA epitopes, antibody MDA3 for MDA epitopes, antibody E06 forOxPL epitopes and a no-antibody control section. The material is stainedwith LA25 for MAA epitopes, antibody MDA3 for MDA epitopes, antibody E06for OxPL epitopes and a LA24 antibody control.

FIG. 22A-D shows pharmacokinetics of ⁸⁹Zr-LA25; radioactivity half-lifeof 29 min, and 13 min for ⁸⁹Zr-LA24 (A). Gamma counting andautoradiography of ⁸⁹Zr-LA24 (grey) and ⁸⁹Zr-LA25 (black) in Apoe^(−/−)mouse aortas (B, C). Biodistribution in Apoe^(−/−) mice (D). ****P<0.0001, * P<0.01.

FIG. 23A-E shows representative coronal fused PET/MR images at 20, 40and 60 min post injection (p.i.) of ⁸⁹Zr-LA25 (top) and ⁸⁹Zr-LA24(bottom) (A). Radioactivity quantification in major organs inatherosclerotic rabbits based on PET/MR imaging (10-60 min), and 24hours p.i. (B). SUV=Standardized uptake values. Pharmacokinetics inatherosclerotic rabbits for ⁸⁹Zr-LA24 and ⁸⁹Zr-LA25, with half-lives of1.1 and 2.2 hours, respectively (C). Ex vivo radioactivity concentration(D) and autoradiography (E) for ⁸⁹Zr-LA24 and ⁸⁹Zr-LA25 in aortas fromrabbits with atherosclerosis 28 hours post injection.

FIG. 24A-F shows representative coronal aortic fused PET/MR imaging 24hours post injection. (A), autoradiography and gamma counting (wholeaortas) 28 hours p.i. of ⁸⁹Zr-LA25 (B). MR T2-weighted imaging (C),¹⁸F-FDG PET/MRI (D), DCE-MRI after (E) and Cy5-rHDL near infraredfluorescence imaging (F). All in healthy control (white) andatherosclerotic rabbit abdominal aortas (black). ****P<0.0001.

FIG. 25 shows digital autoradiography of atherosclerotic rabbit aortasections with adjacent slides stained for H&E, RAM-11 and Oil red 0,with corresponding masks and merged images with autoradiography. On theright Pearson correlations are shown for autoradiography with vesselwall area (r=0.93, P<0.0001), RAM-11 (r=0.74, P=0.0004) and Oil red 0(r=0.70, P=0.0008).

FIG. 26 shows staining of brain tissue for with an antibody of thedisclosure against MAA adducts. The image shows that MAA adducts arestained in glioblastoma multiforme (GBM).

FIG. 27 shows sequences of the VH and VL antibody domains of the variousanti-MAA antibodies of the disclosure. The bolded/underlined sequencecorrespond to CDR domains.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to a “single-chain variablefragment” or “scFv” includes a plurality of single-chain variablefragments and reference to “oxidized phospholipid” includes reference toone or more oxidized phospholipids and equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure. Moreover, withrespect to any term that is presented in one or more publications thatis similar to, or identical with, a term that has been expressly definedin this disclosure, the definition of the term as expressly provided inthis disclosure will control in all respects.

Also, the use of “and” means “and/or” unless stated otherwise.Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Atherosclerosis is a chronic and multifocal inflammatory disease ofmedium and large arteries and the major underlying cause ofcardiovascular disease (CVD). Despite a decline in mortality in theWestern World, the prevalence of CVD has not declined and remains theleading global cause of death. Progression of atherosclerosis is drivenby the accumulation, modification and oxidation of circulating lipids,which drives the influx of immune cells in the vessel wall, leading tochronic inflammation and the development of advanced atheroscleroticplaques. Progressing plaques are prone to develop erosion and/orrupture, resulting in the release of thrombotic material into thecirculation that may lead to luminal occlusion and acute cardiovascularevents, e.g., myocardial infarction, stroke and acute ischemic injury. Alarge subset of such plaques continue to grow until they causemyocardial ischemia, leading to angina pectoris.

Clinical practice relies on detecting ischemia of obstructive lesions todiagnose risk, leaving features like plaque burden and outwardremodeling underappreciated. There is a need for accurate imagingmethods to assess the extent of disease burden and to identify high-risklesions, including those with superficial plaque erosion. Moreover, anemerging paradigm focusing on superficial plaque erosion, departing fromthe classical thrombotic rupture has been suggested.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause ofchronic liver disease in children. NAFLD includes a range of diseasestates from benign steatosis to non-alcoholic steatohepatitis (NASH).The disease may cause cirrhosis with the need for liver transplantationas well as other problems such as metabolic and cardiovascular disease.Although the pathogenesis of NAFLD is still unclear it is likely thatinsulin resistance, increased oxidative stress and lipid peroxidationplay roles. Levels of intracellular glutathione, which protects againstoxidative stress, are low in NAFLD. Two distinct histological forms ofNASH have been described.

Type 1 NASH occurs in adults and some children and is characterized bysteatosis, lobular inflammation, ballooning degeneration andperisinusoidal fibrosis. Type 2 NASH is found most commonly in childrenand is characterized by steatosis, portal inflammation, and portalfibrosis. Schwimmer et al. (Hepatgology, 42(3):641-649, 2005;incorporated herein by reference) described various criteria andbiomarkers used to differentiate NASH Type 1 from NASH Type 2. Inparticular, Schwimmer et al. discloses that subjects with NASH Type 1had higher AST, ALT and triglyceride levels compared to patients withNASH Type 2. However, the strongest factor demonstrating a difference inthe two types of NASH are best found upon histological examination. Asstated above, Type 1 NASH demonstrates a prevalent lobular inflammationin the liver in contrast with a prevalent portal inflammation in Type 2NASH. Thus, the disclosure contemplates that one of the keydifferentiating factors that can be used in the methods disclosed hereinis identifying, by histological examination, the presence of Type 1 vs.Type 2 NASH.

As mentioned non-alcoholic fatty liver disease (NAFLD) represents aspectrum of disease occurring in the absence of alcohol abuse. It ischaracterized by the presence of steatosis (fat in the liver) and mayrepresent a hepatic manifestation of the metabolic syndrome (includingobesity, diabetes and hypertriglyceridemia). The increased generation offree fatty acids for hepatic re-esterification and oxidation results inaccumulation of intrahepatic fat and increases the liver's vulnerabilityto secondary insults. NAFLD is linked to insulin resistance, it causesliver disease in adults and children and may ultimately lead tocirrhosis (Skelly et al., J Hepatol 2001; 35: 195-9; Chitturi et al.,Hepatology 2002; 35(2):373-9). The severity of NAFLD ranges from therelatively benign isolated predominantly macrovesicular steatosis (i.e.,nonalcoholic fatty liver or NAFL) to non-alcoholic steatohepatitis(NASH) (Angulo et al., J Gastroenterol Hepatol 2002; 17 Suppl:S186-90).NASH is characterized by the histologic presence of steatosis,cytological ballooning, scattered inflammation and pericellular fibrosis(Contos et al., Adv Anat Pathol 2002; 9:37-51). Hepatic fibrosisresulting from NASH may progress to cirrhosis of the liver or liverfailure, and in some instances may lead to hepatocellular carcinoma.Because OSEs are inflammatory their presence in liver tissue can lead toincreased inflammation and contribute to the progression of liverdisease including NASH.

For example, NASH subjects have evidence of increased oxidative stressin the liver, often driven by Kupfer cells and non-enzymatic pathways.In addition, NASH subject have a reduced level of IgM antibodies to OSEcompared to normal control (Hendrikxx et al., BMC Med. 14:107, 2016). Inaddition, Bieghs et al. shows that immunization with heat-inactivatedpneumococci, which induce the production of anti-OxLDL antibodies due tomolecular mimicry, led to a reduction in hepatic inflammation inNASH-induced mice (Hepatol., 56(3):894-903, 2012). In addition,protection from MDA epitopes resulted in decreased hepatic inflammationin Ldlr^(−/−) mice fed a western diet and treated with a murine anti-MDAantibody (LR04).

The LDL particle is exquisitely sensitive to oxidative damage due to itscomplex lipid-protein composition and a large number of polyunsaturatedacyl chains. The mechanisms of LDL oxidation in vivo include reactionscatalyzed by 12/15-lipoxygenase (12/15-LO), myeloperoxidase (MPO),nitric oxide synthases and NADPH oxidases, as well as those mediated byheme and hemoglobin (Hb). Small amounts of Hb are constantly leakingfrom damaged erythrocytes, particularly in the vascular regions withturbulent flow, such as arterial bifurcations and aortic curvatures,within the intima of the atrial wall and in vasa vasorum ofatherosclerotic lesions. The presence of OSEs in clinically relevanthuman lesions provides a strong rationale to target such epitopes inplasma and in atherosclerotic plaques for clinical applications.

Oxidation of low-density lipoprotein (LDL), as well as oxidizedphospholipids on apolipoprotein B-100 (OxPL-apoB), which mainly reflectoxidized phospholipids associated with lipoprotein (a), have beenidentified as hallmarks of high cardiovascular risk (see, e.g.,WO2014/018643, the disclosure of which is incorporated herein byreference). When LDL undergoes oxidation, the byproducts of lipidperoxidation generate many pro-inflammatory chemical modifications ofboth the lipid and protein moieties, collectively termedoxidation-specific epitopes (OSEs). Several of these OSEs, such asoxidized phospholipids and malondialdehyde epitopes, are well definedchemically and immunologically. They represent danger-associatedmolecular patterns (DAMPs) and induce a pro-inflammatory response. DAMPsare recognized by the innate immune system via pattern recognitionreceptors, including scavenger receptors IgM natural antibodies andcomplement factor H (CFH), that bind, neutralize and/or facilitate theirclearance. Additionally, prior work has shown that OSEs can be imaged inzebrafish, mice, and rabbit lipid/atherosclerosis models with murine orhuman OSE-targeted antibodies using nuclear and MRI techniques. However,the potential immunogenicity of these approaches may limit clinicalapplication.

Innate natural antibodies (NAbs) provide the first line of host defenseagainst common oxidation-specific epitopes (OSE) on endogenousneo-epitopes (OxLDL and apoptotic cells) and exogenous epitopes ofpathogens, and maintain host homeostasis. OSEs are ubiquitous, formed inmany inflammatory tissues, including atherosclerotic lesions, and are amajor target of IgM NAbs. The prototypic IgM NAb E06, which binds thephosphocholine (PC) headgroup in oxidized phospholipids (OxPL), blocksuptake of OxLDL by macrophages. However, MDA-OSEs are not recognized byE06 and provide the ability for additional diagnostics or therapeuticswith respect to those disease or disorders with more prevalentMDA-related-OSEs.

The term “oxidized LDL” is used to describe a wide variety of LDLpreparations that have been oxidatively modified ex vivo under definedconditions, or isolated from biological sources.

Malondialdehyde (MDA) is a prominent aldehyde product of lipidperoxidation, as well as of eicosanoid metabolism, which can formadducts with the lysine residues of apoB or other proteins. MDA-modifiedLDL has also been isolated and characterized from the plasma of patientswith coronary heart disease. Malondialdehyde-acetaldehyde (MAA) is astable and dominant adduct that can form on various proteins and onOxLDL molecules. MAA adducts form an antigenic epitope recognized byantibodies of the disclosure.

The detection of early forms of oxidized LDL in the plasma has beenfacilitated by the development of monoclonal antibodies (mAbs) specificfor the epitopes of oxidized Apo B or oxidized lipids bound to Apo B.The three well-established mAbs used for immunoassays of oxidized LDLare: (i) FOH1a/DLH3, which was generated by immunizing mice againsthuman coronary atheroma, and which recognizes the phosphorylcholinemoiety of oxidized PC, but not of normal, PC; (ii) 4E6, which wasgenerated by immunizing mice with Cu²⁺-oxidized LDL, and whichrecognizes the MDA-modified lysine epitopes of Apo B; and (iii) E06,which was established from the B cell clones of nonimmunized ApoE-deficient mice, and also recognizes the phosphocholine moiety ofoxidized but not normal PC. MDA-OSEs are not recognized by the mAb E06,which has specificity for PC.

The disclosure provides an antibody and antibody fragment that recognizeMAA-adducts found on various biological molecules including proteins,peptides and, e.g., on MAA-LDL adducts. These antibody and/or antibodyfragments can be used as therapies for fatty liver disease includingNASH as antibodies to OSEs in subjects having fatty liver disease aredecreased compared to normal healthy subjects. Moreover, the antibodyand/or antibody fragments can be used in the diagnosis of diseasesincluding but not limited to atherosclerotic disease and disorders andfatty liver disease and disorders.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity) and may alsoinclude certain antibody fragments. An antibody can be human, humanizedand/or affinity matured.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta), may be performed. Useful effector cells for suchassays include PBMC and NK cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. PNAS (USA)95:652-656 (1998).

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion typically retains at least one, more commonly mostor all, of the functions normally associated with that portion whenpresent in an intact antibody. Examples of antibody fragments includeFab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. In one embodiment, an antibody fragmentcomprises an antigen binding site of the intact antibody and thusretains the ability to bind antigen. In another embodiment, an antibodyfragment, for example one that comprises the Fc region, retains at leastone of the biological functions normally associated with the Fc regionwhen present in an intact antibody, such as FcRn binding, antibodyhalf-life modulation, ADCC function and complement binding. In oneembodiment, an antibody fragment is a monovalent antibody that has an invivo half-life substantially similar to an intact antibody. For example,such an antibody fragment may comprise on antigen binding arm linked toan Fc sequence capable of conferring in vivo stability to the fragment.

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Antigens comprise epitopes that are recognized by antibodies.In a specific embodiment of the disclosure the epitope is amalondialdehyde-acetaldehyde (MAA) adducts. Such MAA adducts may bepresent on a number of biological factors or molecules includingpolypeptides and phospholipids and lipoproteins.

The term “anti-OxPL antibody” or “an antibody that binds to OxPL” refersto an antibody that is capable of binding OxPL with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting OxPL.

The term “anti-MDA-derived-OxPL” or “anti-MAA-derived-OxPL” refers toantibodies that bind to unique epitopes on OxPL that comprise MDA and/orMAA epitopes.

The term “anti-MAA-adduct” refers to an antibody or antibody fragmentthat binds to MAA adducts that can be present on proteins, polypeptides,OxPL and other biological molecules.

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(d)). Affinity can be measured by commonmethods known in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthis disclosure.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The sourceof the biological sample may be solid tissue as from a fresh, frozenand/or preserved organ or tissue sample or biopsy or aspirate; blood orany blood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. In some embodiments,the biological sample is obtained from a primary or metastatic tumor.The biological sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the disclosure. Thisincludes chronic and acute disorders or diseases including thosepathological conditions that predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude cardiovascular disease, atherosclerotic disease and disorders,fatty liver disease including NASH, stenosis, disease and disorders thatare induced by OxLDL, disease and disorders that are induced by MAA oroxidative stress, and diseases and disorders that lead to ischemicinjury due to atherosclerotic plaques.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: Clq bindingand complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “Fc region” as used herein refers to the C-terminal region ofan immunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Such effector functions generally require the Fcregion to be combined with a binding to domain (e.g., an antibodyvariable domain) and can be assessed using various assays as disclosed,for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence that isidentical to the amino acid sequence of an Fc region found in nature.Native sequence human Fc regions include a native sequence human IgG1 Fcregion (non-A and A allotypes); native sequence human IgG2 Fc region;native sequence human IgG3 Fc region; and native sequence human IgG4 Fcregion as well as naturally occurring variants thereof.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one that binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

Fc receptor also includes the neonatal receptor, FcRn, which isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)) and regulation of homeostasis of immunoglobulins. Methods ofmeasuring binding to FcRn are known (see, e.g., Ghetie and Ward.,Immunol. Today 18(12):592-598 (1997); Ghetie et al., NatureBiotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem.279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

“Fv” is the minimum antibody fragment, which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

A Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains have afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409. LR04 is a murine antibody of the disclosure that can behumanized as described herein. In contrast, it will be understood thatLA25, MK17, KA2 and ML7 are human antibodies.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584). See also,for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)regarding human antibodies generated via a human B-cell hybridomatechnology.

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes that mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and typically more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or silver stain. An isolated antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, an isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels, a magnetic metal (e.g.,paramagnetic) or fluorescent labels) or, in the case of an enzymaticlabel, may catalyze chemical alteration of a substrate compound orcomposition which is detectable.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (λ), based on the amino acid sequences of theirconstant domains.

An “MAA-related disease or disorder” refers to a disease or disorderthat is associated or caused by an MAA adduct. Such disease or disordersinclude inflammatory disease or disorders such as, but not limited to,arthersclerotic diseases or disorder, rheumatoid disease or disorder(e.g., rheumatoid arthritis), lung disease or disorder (e.g., resultingfrom toxic inhalants and smoking), cancers (e.g., brain cancer such asglioblastoma, skin cancer, colon cancer, breast cancer, prostate cancer,lung cancer, liver cancer etc.), and liver disease and disordersincluding fatty liver disease, alcoholic liver diseases, non-alcoholicfatty liver disease, non-alcoholic steatohepatitis and the like.Moreover, the MAA-related disease and disorders tend to be related tooxidative stress, which is associated with cardiovascular disease,metabolic syndrome and obesity, autoimmune disease including rheumatoidarthritis, multiple sclerosis, cancer and condition caused by cancertreatment, age related macular degeneration, Alzheimer's disease,senescence, alcoholic liver disease, ischemic reperfusion injury,diabetic nephropathy, nephritis, acute lung injury and inventiondiseases or any infallatory condition associated or caused by theforegoing. The antibodies and antibody fragments of the disclosure canspecifically bind MAA adducts in such disease and disorder and diagnosethe existence of, the prognosis or or treat the disease by inhibitingthe inflammatory mediation of such MAA adducts.

The term cardiovascular diseases, is intended to include but is notlimited to atherosclerosis, acute coronary syndrome, acute myocardialinfarction, myocardial infarction (heart attack), stable and unstableangina pectoris, aneurysms, coronary artery disease (CAD), ischemicheart disease, ischemic myocardium, cardiac and sudden cardiac death,cardiomyopathy, congestive heart failure, heart failure, stenosis,peripheral arterial disease (PAD), intermittent claudication, criticallimb ischemia, and stroke. The term fatty liver disease is intended toinclude non-alcoholic fatty liver disease, steatohepatitis,non-alcoholic steatohepatitis (NASH) and the like.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier term “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody for purposes of this disclosure. In contrast topolyclonal antibody preparations, which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody of a monoclonal antibody preparation is directedagainst a single determinant on an antigen. In addition to theirspecificity, monoclonal antibody preparations are advantageous in thatthey are typically uncontaminated by other immunoglobulins.

The modifier term “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, a monoclonal antibodiesto be used in accordance with the disclosure may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohlerand Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include antibodies wherein the antigen-bindingregion of the antibody is derived from an antibody produced by, e.g.,immunizing macaque monkeys with the antigen of interest. For example,LR04 is a murine antibody. A chimeric LR04 antibody would include (a)the murine VH and VL chains (e.g., or Fab region) while including ahuman Fc region.

“Oligonucleotide,” as used herein, refers to short, typically singlestranded polynucleotides that are generally, but not necessarily, lessthan about 200 nucleotides in length. The terms “oligonucleotide” and“polynucleotide” are not mutually exclusive. The description forpolynucleotides is equally and fully applicable to oligonucleotides.

“Oxidized phospholipids (OxPL)” refer to phospholipids with aphosphocholine (PC) headgroup. OxPL are highly pro-inflammatory andproatherogenic. Phosphocholine, a polar head group on certainphospholipids, has been extensively implicated in cardiovasculardisease. Reactive oxygen species generated during coronary inflammationcauses the oxidation of low density lipoprotein (LDL) to generateoxidized LDL (OxLDL). In fact, cardiovascular diseases (CVD) such asatherosclerosis, unstable angina, or acute coronary syndrome have beenshown to be associated with elevated plasma levels of OxLDL (Itabe andUeda. 2007). LDL is a circulating lipoprotein particle that containslipids with a PC polar head group and proteins, an apoB100 protein.

During oxidation of LDL, PC containing neo-epitopes that are not presenton unmodified LDL are generated. Newly exposed PC on OxLDL is recognizedby scavenger receptors on macrophages, such as CD36, and the resultingmacrophage-engulfed oxLDL proceeds towards the formation ofproinflammatory foam cells in the vessel wall. Oxidized LDL is alsorecognized by receptors on endothelial cell surfaces and has beenreported to stimulate a range of responses including endothelialdysfunction, apoptosis, and the unfolded protein response. PCneo-epitopes are also exposed on LDL following modification withphospholipase A2 or amine reactive disease metabolites, such asaldehydes generated from the oxidation of glycated proteins. Thesealternately modified LDL particles are also pro-inflammatory factors inCVD.

Oxidized phospholipids (OxPL) (phospholipids with a phosphocholine (PC)headgroup) are highly pro-inflammatory and proatherogenic. They arepresent in a wide spectrum of inflammatory diseases, includingatherosclerosis, rheumatoid arthritis, diabetic nephropathy, CNSdiseases including multiple sclerosis, fatty liver diseases includingnon-alcoholic fatty liver disease (NAFLD) and non-alcoholicsteatohepatitis (NASH), and a spectrum of acute and chronic pulmonarydiseases. For example, OxPL are present in the lungs of both mice andhumans infected with a wide variety of viral and bacterial pathogens.OxPL are abundant in bronchial alveolar lavage (BAL) of mice with theseinfections as well as in acute respiratory distress syndrome followingacid installation, or in BAL of mice with COPD secondary to smoking.OxPL are proinflammatory mediators for macrophages, by inducing IL-6 forexample, or alternatively inhibit the capacity of macrophages tophagocytize bacteria. OxPL are prevalent in livers of patients and micewith NASH, and have been shown to be involved in the pathogenesis inmurine models of NASH. OxPL are also extensively present inatherosclerotic lesions, and in vulnerable plaques of human coronaryarteries. They are also released into the circulation duringinterventional procedures such as PCI and stenting, where they likelymediate downstream proinflammatory and vasoactive effects.

Antibodies towards phosphocholine (PC) have been shown to bind oxidized,or otherwise modified, LDL and block the pro-inflammatory activity ofOxLDL in in vivo models or in vitro studies (Shaw et al. 2000; Shaw etal. 2001).

A “polynucleotide,” or “nucleic acid,” as used herein, refer to polymersof nucleotides of any length, and include DNA and RNA. The nucleotidescan be deoxyribonucleotides, ribonucleotides, modified nucleotides orbases, and/or their analogs that can be incorporated into a polymer byDNA or RNA polymerase, or by a synthetic reaction. A polynucleotide maycomprise modified nucleotides, such as methylated nucleotides and theiranalogs. If present, modification to the nucleotide structure may beimparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after synthesis, such as byconjugation with a label. Other types of modifications include, forexample, “caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.). Thepreceding description applies to all polynucleotides referred to herein,including RNA and DNA.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of thedisclosure and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by the values (e.g., K_(d) values). Thedifference between said two values is, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” “substantially increased,” or“substantially different,” as used herein, denotes a sufficiently highdegree of difference between two numeric values (generally oneassociated with a molecule and the other associated with areference/comparator molecule) such that one of skill in the art wouldconsider the difference between the two values to be of statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., K_(d) values). The difference betweensaid two values is, for example, greater than about 10%, greater thanabout 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions orhypervariable regions (CDRs or HVRs, used interchangeably herein) bothin the light-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a (3-sheet configuration, connected bythree HVRs, which form loops connecting, and in some cases forming partof, the (3-sheet structure. The HVRs in each chain are held together inclose proximity by the FR regions and, with the HVRs from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, National Institute of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell and replicate along with the host genome. Moreover,certain vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)—V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

A “variant Fc region” comprises an amino acid sequence, which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, typically one or more amino acid substitution(s).Typically, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and typically from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region of a disclosure possesses atleast about 80% homology with a native sequence Fc region and/or with anFc region of a parent polypeptide, at least about 90% homologytherewith, and typically at least about 95% homology therewith.

This disclosure describes the generation of a monoclonal antibody andantibody fragments directed to oxidation-specific epitopes, specificallymalondialdehyde-acetaldehyde (MAA) epitopes that are involved in avariety of diseases, including cardiovascular disease, liver disease andneurological diseases. The antibody or antibody fragment can be used asa biomarker, molecular imaging agent and therapeutic (“biotheranostic”)in these disease states. This antibody or antibody fragment can be usedas a “passive vaccination” approach to prevent diseases, e.g.,atherosclerosis or liver disease.

The antibodies and antibody fragments of the disclosure were generatedby screening a phage display library for antibodies and then documentingappropriate properties for binding the antigen (MAA). The screeningprocess, antigen selection, its specificity for its target and resultingsequences provides an antibody that is useful for treating humans.

The antibodies of the disclosure can be used in a variety of settings,including immunoassays in plasma in clinical and research assays,immunostaining of tissues, molecular imaging after adding appropriatetags and as an infusion for therapeutic purposes in humans.

The disclosure also provides for single chain variable antibodyfragments (“scFv”), V_(H), V_(L) and complementarity determining regionsthat selectively bind to MAA-OSEs. The scFvs of the disclosure aresoluble and can be readily synthesized. Further, vectors comprisingsequences encoding the scFvs disclosed herein enabled the production ofa transgenic murine model as well as production in a variety of hostcell lines and organisms.

The disclosure provides sequences associated with the light and heavychains or the antibodies of the disclosure (see, FIG. 27 and SEQ IDNos:1-20).

The disclosure provides antibodies, antibody fragments, human andhumanized antibodies that bind to MAA-OSE. Antibody fragments may begenerated by traditional means, such as enzymatic digestion, or byrecombinant techniques. In certain circumstances there are advantages ofusing antibody fragments, rather than whole antibodies. The smaller sizeof the fragments allows for rapid clearance, and may lead to improvedaccess to tumors, plaques and diseased tissue. For a review of certainantibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. Coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage libraries.Alternatively, Fab′-SH fragments can be directly recovered from E. Coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Fab and F(ab′)₂ fragment with increased in vivo half-lifecomprising salvage receptor binding epitope residues are described inU.S. Pat. No. 5,869,046. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In certainembodiments, an antibody is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv arespecies with intact combining sites that are devoid of constant regions;thus, they may be suitable for reduced nonspecific binding during invivo use. scFv fusion proteins may be constructed to yield fusion of aneffector protein at either the amino or the carboxy terminus of an scFv.See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragmentmay also be a “linear antibody”, e.g., as described in U.S. Pat. No.5,641,870, for example. Such linear antibodies may be monospecific orbispecific.

The disclosure, although providing specific antibody sequences andantibody sequence fragments having biological activity, further disclosethat these sequence can be used to generate improved variants.Accordingly, in some instances an antibody or antibody fragment may havea percent identity to the sequences of the disclosure (e.g., 99%-99.9%identity to SEQ ID NO:1-19 or 20).

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis (e.g.,modifications of the sequences set forth in FIG. 27 and SEQ IDNos:1-20). Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion,insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid alterations may be introduced in thesubject antibody amino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu)and replaced by a neutral or negatively charged amino acid (e.g.,Alanine or Polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, Alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.Polyhistidine tags are also useful for purification.

In certain embodiments, an antibody of the disclosure is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn of the CH₂ domain of the Fcregion. See, e.g., Wright et al. (1997) TIBTECH 15:26-32. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

For example, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. Such variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al.), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions that further improve ADCC. Suchsubstitutions may occur in combination with any of the variationsdescribed above.

In certain embodiments, the disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for many applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the antibody are measured to ensurethat only the desired properties are maintained. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the antibody lacks a particularbinding but retains other binding. Non-limiting examples of in vitroassays to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I., et al. Proc. Nat'lAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'lAcad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (seeBruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays mayalso be carried out to confirm that the antibody is unable to bind Clqand hence lacks CDC activity. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, for example, Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Other antibody variants having one or more amino acid substitutions areprovided. Sites of interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions can also be performed. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened, e.g., for a desired activity, such as improvedantigen binding, decreased immunogenicity, improved ADCC or CDC, etc.

The disclosure provides an antibody or antibody fragment capable ofbinding to MAA-OSEs or other MAA related adducts, wherein the antibodyor antibody fragment comprises a variable heavy chain (V_(H)) domainand/or a variable light chain (V_(L)) domain, and wherein (a) the V_(H)domain comprises an amino acid sequence that includes one, two or threecomplementarity determining regions (CDRs) selected from Table 1,wherein only one CDR is selected from each column of CDR1, CDR2 andCDR3; and (b) the V_(L) domain comprises an amino acid sequence thatincludes one, two or three complementarity determining regions (CDRs)selected from Table 1, wherein only one CDR is selected from each columnof CDR1, CDR2 and CDR3.

TABLE 1 Antibody CDR1 CDR2 CDR3 Designation (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) KA2 VH GFTFSSYW (25) INSDGSST (26) CARDYSSSWYFDYW (27)LRO4 VH GYTFTTYG (28) INTYSGVP (29) CAKLGFAYW (30) ML7 VH GFTFSSYG (31)IWYDGSNK (32) CARGSLSGLDVW (33) MK17 VH GFTFSSYG (34) IWYDGSNK (35)CARDRGYPWLRSRGGMDV (36) LA25 VH GFTFSSYG (37) IWYDGSNK (38)CARGRWGGYFDLW (39)

TABLE 2 Antibody CDR1 CDR2 CDR3 Designation (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) KA2 VL QSLLHSNGYNY (40) LGS (41) CMQALQTHSF (42) LRO4 VLKSLLHSNGNTY (43) RMS (44) CMQHLEYPYTF (45) ML7 VL SSNIGSNY (46) SNN (47)CAAWDVSLRQWLF (48) MK17 VL QGIGNY (49) AAS (50) CQQLNGYPLTF (51) LA25 VLSSDVGGYNY (52) EVS (53) CSSYAGSNNYWV (54)

In one embodiment, the antibody or antibody fragment comprises a V_(H)and/or V_(L) domain comprises (a) an amino acid sequence as set forth inSEQ ID NO:2 and/or 4), (b) SEQ ID NO:6 and/or 8, (c) SEQ ID NO:10 and/or12, (d) SEQ ID NO:14 and/or 16, (e) SEQ ID NO:18 and/or 20, (f)sequences that are at least 98-99.9% identical thereto or fragmentsthereof containing at least one CDR from each antibody designation inTable 1 and/or 2 and wherein the antibody or antibody fragment binds toan MAA adduct.

In one embodiment, the disclosure provides an antibody or an scFv withheavy and light chain domains comprising the complementarity determiningregions contained in the amino acid sequences of FIG. 27 (or sequencesthat are 98-99.9% identical thereto). In one embodiment the scFv arelinked to an Fc region.

In one embodiment, the disclosure provides an antibody comprising alight-chain variable region as set forth in SEQ ID NO:2, 6, 10, 14, or18; or a sequence that is 98, 99 or 99.9% identical thereto). In anotherembodiment, the disclosure provides an antibody with a humanized lightchain variable region of SEQ ID NO:10 (LRO4). In another embodiment, thedisclosure provides an antibody that comprises a heavy chain variableregion comprising a sequence as set forth in SEQ ID NO:4, 8, 12, 16 or20; or a sequence that is 98, 99 or 99.9% identical thereto). In anotherembodiment, the disclosure provides an antibody that comprises ahumanized heavy chain variable region of SEQ ID NO:12 (LRO4).

In another embodiment, the disclosure provides a chimeric antibodycomprising, for example, a VH and/or VL of SEQ ID NO:10 and/or 12,respectively, and a human Fc region.

In one embodiment, the disclosure provides an scFv comprising a linkerbetween the light change variable region and the heavy-chain variableregion. The linker can be any number of commonly used peptide linkers.In one embodiment, the linker comprises a repeating unit of GGGS (SEQ IDNO:57). The repeat of GGGS may be 2, 3, 4 or more times.

In another embodiment, the disclosure comprises a scFv comprising alight chain variable region as set forth in FIG. 27 (e.g., SEQ ID NO:2,6, 10, 14 or 18) linked by a peptide linker to it's corresponding heavychain variable region as set forth in FIG. 27 (e.g., SEQ ID NO:4, 8, 12,16 or 20), respectively. In a further embodiment, the disclosureprovides for an scFv that has a polypeptide sequence that has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, at least 99% or at least 99.9% sequence identity to the sequencesset forth in FIG. 27 and which selectively binds to an MAA-OSE on anoxidized phospholipid.

Nucleic acid molecules encoding the amino acid sequences of theantibodies, antibody fragments and variants of the antibody are preparedby a variety of methods known in the art. Nucleic acid coding sequencesfor the antibodies and antibody fragments described herein are providedin FIG. 27 and SEQ ID Nos:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.

In a particular embodiment, the disclosure provides for a scFv which isencoded by a polynucleotide sequence provided in FIG. 27 (e.g., SEQ IDNO:1 and/or 3; SEQ ID NO:5 and/or 7; SEQ ID NO:9 and/or 11; SEQ ID NO:13and/or 15; or SEQ ID NO:17 and/or 19). In a further embodiment, thedisclosure provides for a scFv which is encoded by a polynucleotidesequence that has at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, at least 99% or at least 99.9% sequenceidentity to the sequences in FIG. 27 and which produces a polypeptidesthat selectively binds to MAA-OSEs.

The disclosure also encompasses humanized antibodies containing sequencefrom SEQ ID NO:10 and 12 (LRO4). Various methods for humanizingnon-human antibodies are known in the art. For example, a humanizedantibody can have one or more amino acid residues introduced into itfrom a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody. See, e.g., Sims et al.(1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol.196:901. Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. See, e.g., Carter et al. (1992) Proc.Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol.,151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

The disclosure further provides for a scFv disclosed herein that furthercomprises a fragment crystallizable region (“Fc”) of an antibody. In aparticular embodiment, the Fc region is from a human or humanizedantibody. The Fc region is the tail region of an antibody that interactswith cell surface receptors called Fc receptors and some proteins of thecomplement system. This property allows antibodies to activate theimmune system. In IgG, IgA and IgD antibody isotypes, the Fc region iscomposed of two identical protein fragments, derived from the second andthird constant domains of the antibody's two heavy chains; IgM and IgEFc regions contain three heavy chain constant domains (C_(H) domains2-4) in each polypeptide chain. The Fc regions of IgGs bear a highlyconserved N-glycosylation site. Glycosylation of the Fc fragment isessential for Fc receptor-mediated activity. The N-glycans attached tothis site are predominantly core-fucosylated diantennary structures ofthe complex type. In addition, small amounts of these N-glycans alsobear bisecting GlcNAc and α-2,6 linked sialic acid residues. The otherpart of an antibody, called the Fab region, contains variable sectionsthat define the specific target that the antibody can bind. The scFv ofthe disclosure are comprised of elements from the Fab region. Bycontrast, the Fc region of all antibodies in a class are the same foreach species; they are constant rather than variable. The Fc region is,therefore, sometimes termed the “fragment constant region”. Accordingly,the polynucleotide and polypeptide sequences which encode the Fc regionsfor countless species have already been determined and would be known byone of skill in the art.

Polynucleotide sequences encoding polypeptide components of the antibodyor antibody fragments of the disclosure can be obtained using standardrecombinant techniques. Desired polynucleotide sequences may be isolatedand sequenced from antibody producing cells such as hybridoma cells.Alternatively, polynucleotides can be synthesized using nucleotidesynthesizer or PCR techniques. Once obtained, sequences encoding thepolypeptides are inserted into a recombinant vector capable ofreplicating and expressing heterologous polynucleotides in prokaryotichosts. Many vectors that are available and known in the art can be usedfor the purpose of the present invention. Selection of an appropriatevector will depend mainly on the size of the nucleic acids to beinserted into the vector and the particular host cell to be transformedwith the vector. Each vector contains various components, depending onits function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides. The vector components generally include, butare not limited to: an origin of replication, a selection marker gene, apromoter, a ribosome binding site (RBS), a signal sequence, theheterologous nucleic acid insert and a transcription terminationsequence.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizinga nucleotide sequence for expression in a plant is provided, forexample, in U.S. Pat. No. 6,015,891, and the references cited therein.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of nucleic acids differing intheir nucleotide sequences can be used to encode a given antibody of thedisclosure.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. Coli istypically transformed using pBR322, a plasmid derived from an E. Colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage vectors may be utilized in making a recombinant vectorwhich can be used to transform susceptible host cells such as E. ColiLE392.

The expression vector of the disclosure may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the (3-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one embodiment, each cistron within the recombinant vector comprisesa secretion signal sequence component that directs translocation of theexpressed polypeptides across a membrane. In general, the signalsequence may be a component of the vector, or it may be a part of thetarget polypeptide DNA that is inserted into the vector. The signalsequence selected for the purpose of this invention should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe signal sequences native to the heterologous polypeptides, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signalsequences used in both cistrons of the expression system are STII signalsequences or variants thereof.

In another embodiment, the production of the immunoglobulins accordingto the disclosure can occur in the cytoplasm of the host cell, andtherefore does not require the presence of secretion signal sequenceswithin each cistron. In that regard, immunoglobulin light and heavychains are expressed, folded and assembled to form functionalimmunoglobulins within the cytoplasm. Certain host strains (e.g., the E.Coli trxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies of thedisclosure include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. Coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. Coli cells are used as hosts forthe disclosure. Examples of E. Coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 (U.S. Pat.No. 5,639,635). Other strains and derivatives thereof, such as E. Coli294 (ATCC 31,446), E. Coli B, E. ColiX 1776 (ATCC 31,537) and E. ColiRV308 are also suitable. These examples are illustrative rather thanlimiting. Methods for constructing derivatives of any of theabove-mentioned bacteria having defined genotypes are known in the artand described in, for example, Bass et al., Proteins, 8:309-314 (1990).It is generally necessary to select the appropriate bacteria taking intoconsideration replicability of the replicon in the cells of a bacterium.For example, E. Coli, Serratia, or Salmonella species can be suitablyused as the host when well-known plasmids such as pBR322, pBR325,pACYC177, or pKN410 are used to supply the replicon.

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the disclosure aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol. The prokaryotic host cells are cultured at suitabletemperatures.

In one embodiment, the expressed polypeptides are secreted into andrecovered from the periplasm of the host cells. Protein recoverytypically involves disrupting the microorganism, generally by such meansas osmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins can be transported into theculture media and isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

Large scale or small scale fermentation can be used and can be optimizedusing skills well known in the art.

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration.

The disclosure further provides for an expression vector which encodes ascFv or humanized antibody disclosed herein that is transferred into asuitable host organism. The suitable host organism is a microorganism,yeast or a mammalian cell system. Typically, the mammalian cell systemis monocyte-derived (e.g., macrophages, monocytes, and neutrophils),lymphocyte-derived (e.g., myeloma, hybridoma, and a normal immortalizedB cell), parenchymal (e.g., hepatocytes) and non-parenchymal cells(e.g., stellate cells).

Additionally, the disclosure also provides for a unique transgenicanimal model that expresses a scFv disclosed herein from both the liverand from macrophages. This animal model allows for a systematic study ofthe therapeutic effects of the scFvs of the disclosure in a wide varietyof physiological and pathophysiological settings. The methods andcompositions presented herein can be equally as well be applied tocreate transgenic models in any number of animals including, but notlimited to, rats, rabbits, pigs, sheep, goats, and horses. Thedisclosure, therefore, provides methods that can be performed in vivo tostudy the therapeutic possibilities of a scFv of the disclosure or anantibody of the disclosure in a highly defined manner. For example, adesirable scFv can be produced during cell culturing or in a transgenicanimal. The availability of a transgenic animal model expressing a scFvdisclosed herein allows for in-depth preclinical testing for myriad ofpotential applications. For example, interventions can be done on thetransgenic animals to test the impact of scFv expression, including, bybreeding the animals into a variety of backgrounds.

The antibody and antibody fragments disclosed herein bind to MAA adductsand can block their pro-inflammatory effects. Such proinflammatoryeffects include MAA-related disease and disorders and are relevant to,for example, cardiovascular disease, artherosclerosis, rheumatoidarthritis, lung tissue injury (e.g., caused by smoking), brain lesions,apoptosis, senescence and fatty liver disease (e.g., NASH). The in vivouse of an antibody fragment (humanized, human and non-humanized) of thedisclosure or a human, humanized and non-human antibody of thedisclosure can be used to (a) block inflammation from MAA adducts, (b)treat any one or more of cardiovascular disease, arthersclerosis,rheumatoid arthritis, lung tissue injury (e.g., cause by smoking), brainlesions, apoptosis, senescence and fatty liver disease (e.g., NASH) byblocking the inflammatory effects of MAA adducts, (c) detect and/ordiagnose inflammatory disease or disorders by detecting MAA adducts in asample from or tissue in a subject. For example, the antibody orantibody fragments of the disclosure can be used to treatatherosclerotic diseases and disorders and fatty liver disease includingNASH. In addition, the antibody or antibody fragments of the disclosurecan be labeled and used as diagnostics wherein their binding can beimaged or analyzed to determine the location and/or severity of MAAadducts. Such diagnostics can be used to determine the presence ofatherosclerotic disease and disorders as well as fatty liver diseaseincluding NASH etc.

An embodiment of the disclosure is a method of treating NASH or NAFLD ina patient in need of such therapy, with a human, or humanized antibodytargeting MAA, or fragment thereof, wherein the treatment decreasesliver fat, or inflammation or disease. Such a decrease in liver fat canbe measured, e.g., by magnetic resonance imaging-proton density fattyfraction (MRI-PDFF). An embodiment of the disclosure is a method oftreating NASH or NAFLD in a patient in need of such therapy, with ahuman, or humanized antibody targeting MAA, or fragment thereof asdisclosed herein, wherein the treatment decreases liver fibrosis asmeasured by magnetic resonance elastography (MRE) and/or decreasesinflammation and/or provide a return of liver function biomarkers towithin normal ranges. In another embodiment, the one or more markers ofliver function are selected from the group consisting of alanineaminotransferase (ALT), alkaline phosphatase (ALP), aspartateaminotransferase (AST), gamma-glutamyl transpeptidase (GGT),triglycerides, and lipoproteins (e.g., LDL). In a further embodiment, anALT level of about 60-150 units/liter is indicative of fatty liverdisease. In yet another or further embodiment, an ALP level of about150-250 units/liter is indicative of fatty liver disease. In yet anotherof further embodiment, an AST level of about 40-100 units/liter isindicative of fatty liver disease. In still another or furtherembodiment, a GGT level of 50-100 units/liter is indicative of fattyliver disease. In still another of further embodiment, a triglyceridelevel above 150 mg/dL and/or high LDL level is indicative of fatty liverdisease. In yet another or further embodiment, a resistin level ofgreater than 8 ng/ml is indicative of fatty liver disease. In still yetanother or further embodiment, an adiponectin level decreased by atleast about 20% from age and sex matched normal subjects is indicativeof fatty liver disease. An effective therapy using an antibody orfragment of the disclosure would decrease the leves of AST to between 10to 40 units/L, ALT to between 7 and 56 units/L, ALP to between 44 and147 units/L etc. An embodiment of the disclosure is a method of treatingNASH or NAFLD in a patient in need of such therapy, with a human, orhumanized antibody targeting MAA, or fragment thereof, wherein thetreatment decreases liver fibrosis as measured by MRE or decreases liverfat as measured MRI-PDFF accompanied by an improvement in liver functionas measured by ALT, AST, AST/ALT ratios, bilirubin or GGT. In oneembodiment of the disclosure, a method of treating NASH or NAFLD in apatient in need of such therapy, with a human, or humanized antibodytargeting MAA, or fragment thereof, wherein the treatment decreasesliver fibrosis as measured by MRE or decreases liver fat as measuredMRI-PDFF accompanied by an improvement in insulin sensitivity, forexample, as measured by HbA1c.

The antibodies, antibody fragments and polypeptides of the disclosuremay be used to inhibit or reduce the formation of a senescent cell in aclinically significant or biologically significant manner. As discussedin detail herein, the antibodies, antibody fragments and polypeptides ofthe disclosure are used in an amount and for a time sufficient thatprohibit or reduce the formation of senescent cells in a clinicallysignificant or biologically significant manner. The antibodies, antibodyfragments and polypeptides of the disclosure can prohibit or reduce theformation of one or more types of senescent cells (e.g., senescentpreadipocytes, senescent endothelial cells, senescent fibroblasts,senescent neurons, senescent epithelial cells, senescent mesenchymalcells, senescent smooth muscle cells, senescent macrophages, orsenescent chondrocytes).

A senescent cell may exhibit any one or more of the following sevencharacteristics. (1) Senescence growth arrest is essentially permanentand cannot b e reversed by known physiological stimuli. (2) Senescentcells increase in size, sometimes enlarging more than twofold relativeto the size of non-senescent counterparts. (3) Senescent cells express asenescence-associated b-galactosidase (SAP-gal), which partly reflectsthe increase in lysosomal mass. (4) Most senescent cells expresspl6INK4a, which is not commonly expressed by quiescent or terminallydifferentiated cells. (5) Cells that senesce with persistent DDRsignaling harbor persistent nuclear foci, termed DNA segments withchromatin alterations reinforcing senescence (DNA-SCARS). These focicontain activated DDR proteins and are distinguishable from transientdamage foci. DNA-SCARS include dysfunctional telomeres or telomeredysfunction-induced foci (TIF). (6) Senescent cells express and maysecrete molecules associated with senescence, which in certain instancesmay be observed in the presence of persistent DDR signaling, which incertain instances may be dependent on persistent DDR signaling for theirexpression. (7) The nuclei of senescent cells lose structural proteinssuch as Lamin B 1 or chromatin-associated proteins such as histones andHMGB1. See, e.g., Freund et al, Mol. Biol. Cell 23:2066-75 (2012);Davalos et al, J. Cell Biol. 201:613-29 (2013); Ivanov et al, J. CellBiol. DOI:10. 1083/jcb. 2012121 10, page 1-15; published online Jul. 1,2013; Funayama et al, J. Cell Biol. 175:869-80 (2006)).

Senescent cells and senescent cell associated molecules can be detectedby techniques and procedures described in the art. For example, thepresence of senescent cells in tissues can be analyzed by histochemistryor immunohistochemistry techniques that detect the senescence marker,SA-beta galactosidase (SA-Pgal) (see, e.g., Dimri et al, Proc. Natl.Acad. Sci. USA 92: 9363-9367 (1995)). The presence of the senescentcell-associated polypeptide pl6 can be determined by any one of numerousimmunochemistry methods practiced in the art, such as immunoblottinganalysis. Expression of pl6 mRNA in a cell can be measured by a varietyof techniques practiced in the art including quantitative PCR. Thepresence and level of senescent cell associated polypeptides (e.g.,polypeptides of the SASP) can be determined by using automated and highthroughput assays, such as an automated Luminex array assay described inthe art (see, e.g., Coppe et al., PLoS Biol 6: 2853-68 (2008)).

The presence of senescent cells can also be determined by detection ofsenescent cell-associated molecules, which include growth factors,proteases, cytokines (e.g., inflammatory cytokines), chemokines,cell-related metabolites, reactive oxygen species (e.g., H₂O₂),oxidation specific epitopes, such as MAA, and other molecules thatstimulate inflammation and/or other biological effects or reactions thatmay promote or exacerbate the underlying disease of the subject.Senescent cell-associated molecules include those that are described inthe art as comprising the senescence-associated secretory phenotype(SASP, i.e., which includes secreted factors which may make up thepro-inflammatory phenotype of a senescent cell), senescent-messagingsecretome, and DNA damage secretory program (DDSP). These groupings ofsenescent cell associated molecules, as described in the art, containmolecules in common and are not intended to describe three separatedistinct groupings of molecules. Senescent cell-associated moleculesinclude certain expressed and secreted growth factors, proteases,cytokines, and other factors that may have potent autocrine andparacrine activities (see, e.g., Coppe et al., supra; Coppe et al. J.Biol. Chem. 281:29568-74 (2006); Coppe et al., PLoS One 5:39188 (2010);Krtolica et al. Proc. Natl. Acad. Sci. U.S.A. 98:12072-77 (2001);Parrinello et al., J. Cell Sci. 118:485-96 (2005). ECM associatedfactors include inflammatory proteins and mediators of ECM remodelingand which are strongly induced in senescent cells (see, e.g., Kuilman etal., Nature Reviews 9:81-94 (2009)). Other senescent cell-associatedmolecules include extracellular polypeptides (proteins) describedcollectively as the DNA damage secretory program (DDSP) (see, e.g., Sunet al., Nature Medicine 18:1359-1368 (2012)). Senescent cell-associatedproteins also include cell surface proteins (or receptors) that areexpressed on senescent cells, which include proteins that are present ata detectably lower amount or are not present on the cell surface of anonsenescent cell.

Senescence cell-associated molecules include secreted factors which maymake up the pro-inflammatory phenotype of a senescent cell (e.g., SASP).These factors include, without limitation, GM-CSF, GROa, GRC-a,b,g,IGFBP-7, IL-1a, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1a, MMP-1, MMP-10,MMP-3, Amphiregulin, ENA-78, Eotaxin-3, GCP-2, GITR, HGF, ICAM-1,IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-I b, MCP-4, MIF, MIP-3a,MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, osteoprotegerin, PIGF,RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, angiogenin, Axl, bFGF, BLC,BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, 1-309, IFN-g,IGFBP-1, IGFBP-3, IL-1 Rl, IL-1 1, IL-15, IL-2R-a, IL-6 R, ITAC, Leptin,LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII,Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-b3,MIP-1-delta, IL-4, FGF-7, PDGF-BB, IL-1 6, BMP-4, MDC, MCP-4, IL-10,TIMP-, Fit-3 Ligand, ICAM-1, Axl, CNTF, INF-g, EGF, BMP-6. Additionalidentified factors, which include those sometimes referred to in the artas senescence messaging secretome (SMS) factors, some of which areincluded in the listing of SASP polypeptides, include withoutlimitation, IGF1, IGF2, and IGF2R, IGFBP3, IDFBP5, IGFBP7, PA11, TGF-b,WNT2, IL-la, IL-6, IL-8, and CXCR2-binding chemokines. Cell-associatedmolecules also include without limitation the factors described in Sunet al, Nature Medicine, supra, and include, including, for example,products of the genes, MMP1, WNT16B, SFRP2, MMP12, SPINK1, MMP10, ENPP5,EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14,NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2, TMEM155, EDN1, PSG9,ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15,C17orf67, CALCA, FGF18, IL8, BMP2, MATN3, TFP1, SERPINI 1, TNFRSF25, andIL23A. Senescent cell-associated proteins also include cell surfaceproteins (or receptors) that are expressed on senescent cells, whichinclude proteins that are present at a detectably lower amount or arenot present on the cell surface of a non-senescent cell.

In certain embodiments, the antibodies, antibody fragments andpolypeptides of the disclosure are capable of prohibiting, inhibiting orreducing the formation of at least senescent preadipocytes may be usefulfor treatment of diabetes (particularly type 2 diabetes), metabolicsyndrome, or obesity. In other embodiments, the antibodies, antibodyfragments and polypeptides of the disclosure are capable of prohibitingor reducing the formation of at least senescent endothelial cells,senescent smooth muscle cells, and/or senescent macrophages. Theantibodies, antibody fragments and polypeptides of the disclosure may beuseful for treatment of a cardiovascular disease (e.g.,atherosclerosis). In other particular embodiments, the antibodies,antibody fragments and polypeptides of the disclosure are capable ofprohibiting or reducing the formation of at least senescent fibroblasts.In still another embodiment, the antibodies, antibody fragments andpolypeptides of the disclosure are capable of prohibiting or reducingthe formation of at least senescent neurons, includingdopamine-producing neurons. In still another embodiment, the antibodies,antibody fragments and polypeptides of the disclosure are capable ofprohibiting or reducing the formation of at least senescent retinalpigmented epithelial cells or other senescent epithelial cells (e.g.,pulmonary senescent epithelial cells orsenescent kidney (renal)epithelial cells). Prohibiting or reducing the formation of at leastsenescent pulmonary epithelial cells may be useful for treatingpulmonary diseases, such as chronic obstructive pulmonary disease oridiopathic pulmonary fibrosis. In yet other embodiments, the antibodies,antibody fragments and polypeptides of the disclosure are capable ofprohibiting or reducing the formation of at least senescent immune cells(such as senescent macrophages). In still another embodiment, theantibodies, antibody fragments and polypeptides of the disclosure arecapable of prohibiting or reducing the formation of at least senescentchondrocytes, which may be useful for treatment of an inflammatorydisorder, such as osteoarthritis.

Thus, the antibodies, antibody fragments and polypeptides of thedisclosure can be used to treat inflammatory diseases and disorders,cardiovascular diseases, liver diseases and disorder (e.g., NASH) anddiseases associated with oxidative stress and damage.

For diagnostic applications, the antibody or antibody fragments of thedisclosure will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹³¹I; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; a magnetic or paramagnetic element or compound,or an enzyme, such as alkaline phosphatase, beta-galactosidase orhorseradish peroxidase. In some embodiments, therapeutic or diagnosticradioisotopes or other labels (e.g., PET or SPECT labels) can beincorporated in the agent for conjugation to the EGFRvIII antibodies asdescribed herein. Examples of a radioisotope or other labels include,but are not limited to, ¹¹C, ¹³N, ¹⁵N, ¹⁵O, ³⁵B, ¹⁸F, ³³P, ⁴⁷Sc, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁶Br, ⁷⁷Br, ⁸⁶Y,⁸⁹Zr, ⁹⁰Y, ⁹⁴Tc, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Tc, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd,¹¹¹Ag, ¹¹¹In, ¹¹³In, ¹²¹Te, ¹²²Te, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁵Te ¹²⁶I, ¹³¹In,¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁵³Pb, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶H, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁶⁹Re, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰¹Tl,²⁰³Hg, ²¹¹At, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁴Ac, or ²²⁵Ac.

Any method known in the art for conjugating the antibody or fragment tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies or fragment thereof of the disclosure also are useful forin vivo imaging, wherein an antibody labeled with a detectable moietysuch as a magnetic, paramagnetic, radio-opaque agent or radioisotope isadministered to a subject, typically into the bloodstream, and thepresence and location of the labeled antibody in the host is assayed.This imaging technique is useful in identifying oxidized phospholipiddeposition for the determination of fatty liver disease, NASH,atherosclerotic plaques and cardiovascular diseases or disorders. Suchmethods can be used to determine the existence of a disease and/or tofollow the disease progression and treatment (e.g., imaging before andthen after a round of treatment to determine a therapeutic effect and/orprogression of the disease). The antibody may be labeled with any moietythat is detectable in a host, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, a scFv (humanized or non-humanized) of the disclosureor a humanized antibody of the disclosure are used to delay developmentof a disease or disorder.

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is a human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of thedisclosure, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

Therapeutic and/or diagnostic formulations/preparations comprising anantibody or fragment thereof of the disclosure are prepared for storageby mixing the antibody or fragment having the desired degree of puritywith optional physiologically acceptable carriers, excipients orstabilizers (Remington: The Science and Practice of Pharmacy 20thedition (2000)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Anti-MAA-OSE antibodies of the disclosure can be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, anti-MAA-adductantibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957, incorporated herein by reference.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding ananti-MAA-adduct antibody or fragment thereof of the disclosure into theanimal or plant by standard transgenic techniques. See Hogan and U.S.Pat. No. 6,417,429. The transgenic cells used for making the transgenicanimal can be embryonic stem cells or somatic cells or a fertilized egg.The transgenic non-human organisms can be chimeric, nonchimericheterozygotes, and nonchimeric homozygotes. See, e.g., Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold SpringHarbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: APractical Approach, Oxford University Press (2000); and Pinkert,Transgenic Animal Technology: A Laboratory Handbook, Academic Press(1999), all incorporated herein by reference. In some embodiments, thetransgenic non-human animals have a targeted disruption and replacementby a targeting construct that encodes a heavy chain and/or a light chainof interest. In another embodiment, the transgenic animals comprise andexpress nucleic acid molecules encoding heavy and light chains thatspecifically bind to MAA epitopes or other MAA adducts. In someembodiments, the transgenic animals comprise nucleic acid moleculesencoding a modified antibody such as a single-chain antibody, a chimericantibody or a humanized antibody. The antibodies may be made in anytransgenic animal. In another embodiment, the non-human animals aremice, rats, sheep, pigs, goats, cattle or horses. The non-humantransgenic animal expresses said encoded polypeptides in blood, milk,urine, saliva, tears, mucus and other bodily fluids.

The following examples are intended to illustrate but not limit thedisclosure. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

Cloning and Characterization of LA25.

Human LDL was freshly isolated from plasma of healthy donors after anovernight fast by sequential ultra-centrifugation. LDL (or bovine serumalbumin (BSA)) was modified with MAA, MDA or CuSO₄ to generate MAA-LDL,MAA-BSA, MDA-LDL or copper-oxidized LDL (Cu-OxLDL) respectively. In MAAand MDA preparations, >90% of the lysines were modified as judged usingthe trinitrobenzenesulfonic acid assay.

To enrich for antibodies, a library (κ/λ) was constructed fromlymphocytes of umbilical cord blood of 7 newborn babies and used in thefirst round of screening. The selecting epitope was MAA-BSA. MAA is aspecific advanced MDA-type adduct that has been shown as animmunodominant MDA epitope. After 4 rounds of panning with each library,an enrichment in the newborn library of fully or very near germlinesequences, consistent with minimal non-templated insertions in the CDR3region was obtained. The resultant Fabs were cloned into the phagemidpComb3X. LA25 was identified as a leading candidate due to itsspecificity for MAA epitopes and optimal expression characteristics.LA24 was identified from the same library, but did not bind any relevantoxidation-specific epitopes present in vivo. Specificity of individualphage Fab and soluble Fab were assessed by ELISA. The specificity of Fabbinding was further determined by competition chemiluminescent ELISA anddata expressed as B/B_(o), where B represents binding in the presenceand B_(o) in the absence of competitor. High-titer clone LA25 andcontrol LA24 were selected and converted to the plasmid producingsoluble Fab. Nucleotide and amino acid sequences of Fab clones werecompared to those contained in public databases by using Ig-BLAST andIMGT/VQUEST. LA25 and LA24 plasmid from selected Fab phage clones werecodon optimized and transformed into E. coli C41 (DE3) (Lucigen) forproduction of soluble Fab.

Mouse and Rabbit Atherosclerosis Models.

Female Apoe^(−/−) mice (B6.129P2-Apoe^(tmlUnc)/J, 8-10 weeks old) werepurchased from the Jackson Laboratory (Bar Harbor, Me.) and fed aWestern diet (Harlan Teklad TD.88137, 42% calories from fat) for 22weeks. Male New Zealand White rabbits (2.5-3.0 months old) werepurchased from Charles River Laboratories (Wilmington, Mass.). Topromote the formation of atherosclerotic plaques, endothelial denudationof the aorta was performed after intramuscular (i.m.) administration ofketamine (20 mg/kg) (Fort Dodge Animal Health, Overland Park, Kans.,USA) and Xylazine (5 mg/kg) (Bayer AG, Leverkusen, Germany). A4F-Fogarty embolectomy catheter (Edwards Lifesciences, Irvine, Calif.)was inflated at the level of the left subclavian artery and slowlydeflated while retracting until the iliac bifurcation, under X-rayguidance (Philips Allura Xper FD20/10, Philips Healthcare, Best, TheNetherlands). The procedure was repeated using the contralateral femoralartery as point of entry 4 weeks after the first procedure, i.e., 6weeks after the initiation of a high cholesterol diet (regular chow dietenriched with 0.3% cholesterol, 4.7% coconut oil, Research diets, Inc.,Brunswick, N.J.). After 8 weeks, diet was changed to a 0.15% enrichedcholesterol diet and continued for the remaining 2.5 months before theexperiments were performed. Untreated healthy rabbits were fed a regularchow diet and age-matched to serve as controls.

All animal experiments were performed in accordance with protocolsapproved by the Institutional Animal Care and Use Committees of theinstitution and followed National Institutes of Health guidelines foranimal welfare.

Immunostaining of Human Specimens.

Hearts of patients who had died suddenly with coronary artery diseasewere obtained. Cases were identified retrospectively by the presence ofearly and late fibroatheroma, thin cap fibroatheroma and plaque rupture.Formalin-fixed, paraffin embedded coronary segments were cut at 5-μmthick sections, mounted on charged slides, and stained with hematoxylinand eosin (H&E) or the modified Movat pentachrome method.

Paraffin embedded tissue sections were deparaffinized with Histoclear®,rehydrated through graded ethanol, and blocked with 5% normal goatserum/1% BSA/TBS for 30 minutes. Tissues were incubated overnight at 4°C. with the human monoclonal antibody Fab LA25 or LA24 diluted withblocking buffer. Using the avidin-biotin-alkaline phosphatase method,tissues were incubated for 30 minutes with biotinylated monoclonalanti-HA antibody diluted with blocking buffer in a ratio of 1:1000(Sigma B9183). Tissues were then incubated with ABC Alkaline phosphatasereagent (Vector AK-5000) for 30 minutes and visualized with Vector Redsubstrate (Vector SK-5100). Finally, slides were then counterstainedwith hematoxylin for 30 seconds, dehydrated through graded ethanol,cleared with Histoclear and a coverslip was adhered using histomount.Immunostaining of adjacent sections in the absence of primary Fab wereused as negative controls.

Distal protection devices were obtained from 24 patients undergoingclinically indicated coronary and peripheral procedures. At the end ofthe procedure, the recovered filters from the distal protection deviceswere placed in ice-cold phosphate buffered solution containing EDTA/BHT(4 μM/20 μM). The filter bottom was cut off, the filter inverted and thematerial was paraffin embedded en bloc. Filters were chosen that hadvisible (yellow/white) material at bottom of filter. Seven μm serialsections were prepared, rehydrated, and immunostained with antibodiesLA25 and LA24. The collection of materials was approved by the UCSDHuman Research Subjects Protection Program.

Radiolabeling of Fab LA25 and Non-Specific Isotype Control Fab LA24.

Modification of LA24 and LA25 for labeling with ⁸⁹Zr was carried.Briefly, to a solution of LA24 or LA25 Fab (1-2 mg/ml) in 0.1 Mcarbonate buffer, pH 8.7-8.9, was addedp-isothiocyanatobenzyl-desferrioxamine (Macrocyclics, Plano, Tex.; 5mg/ml in DMSO) until a 2:1 mol ratio was achieved. The mixture wasreacted at 37° C. for 2 h, then allowed to cool down to roomtemperature. The DFO-modified Fab was purified by centrifugal filtrationusing 10 kDa molecular weight cut-off (MWCO) filter tubes and washingtwice with ample fresh PBS to prevent excessive concentration. Forradiolabeling, the DFO-bearing Fabs were reacted with [⁸⁹Zr] Zirconium(IV) oxalate in PBS pH 7.1-7.4 for 2 h. After cooling down, theradiolabeled Fabs were purified by gel filtration using PD-10 columnsand PBS as eluent. The radiochemical yield was 86±9% (n=5), and theradiochemical purity 96±9% (n=5), as assessed by size exclusionchromatography. The specific activities were 2.7±0.1 mCi/mg (n=2) and3.8±0.4 mCi/mg (n=3) for ⁸⁹Zr-LA24 or ⁸⁹Zr-LA25, respectively.

Pharmacokinetics and Biodistribution of ⁸⁹Zr-LA24 and ⁸⁹Zr-LA25 in Mice.

Pharmacokinetic profile and biodistribution evaluation of the PET tracer⁸⁹Zr-LA25 was carried out in Apoe^(−/−) mice (n=13, mean weight 30.8±6.8g), using ⁸⁹Zr-LA24 as chemical control. Radioactivity half-life wasdetermined in blood after lateral tail-vein injection. Animals wereinjected with ⁸⁹Zr-LA25 or ⁸⁹Zr-LA24 (23±1 μCi, 6-8 μg). Blood was drawnfrom the tail vein at 1, 30, 60, 120, 180, and 240 minutes afterinjection. Blood was weighted and counted using a Wizard² 2480 automaticgamma counter (Perkin Elmer, Waltham, Mass.). At 4 hours afterinjection, all mice were sacrificed using an overdose of Isofluorane(Baxter, Deerfield, Ill.) and perfused through the heart with 20 mlsaline. The following organs were harvested: brain, heart, lung, spleen,liver, kidneys, skeletal muscle and bone were harvested and weightedbefore counting using a Wizard² 2480 automatic gamma counter.Radioactivity concentration in tissues was calculated as percentage ofinjected dose per gram (% ID/g).

Immunofluorescence of Mouse Aortic Roots.

Aortic mouse roots were harvested 4 hours after injection, put inoptimal cutting temperature (OCT) and cut in 7 μm thick sections. Thefirst slide was subjected to autoradiography, while adjacent sectionswere stained for cell nuclei (DAPI, blue), macrophages (CD68, red) andendothelial cells (CD3l, green). All antibodies for immunofluorescencestaining were ordered from Bio-Rad, Herculus, Calif., USA.

Immunostaining of Mouse and Rabbit Livers.

Rabbit and mouse livers were harvested and put in formalin beforere-embedding in paraffin <72 hours after harvest. Animal liver specimenswere subsequently stained according to the same protocol as describedfor human specimen.

Pet/MR Imaging.

Rabbits (n=12, mean weights: 3.4±0.9 kg for rabbits withatherosclerosis, and 3.2±0.1 kg for healthy control rabbits). A24G-catheter was introduced in the marginal ear vein for injection witheither ⁸⁹Zr-LA25 or ⁸⁹Zr-LA24 (0.94±0.22 mCi, 0.3-0.4 mg). In thecontralateral ear, a 22G-catheter was used for the administration of thegadolinium based contrast agent; gadopentetate dimeglumine (Magnevist,Bayer Healthcare). Anesthesia was induced by intramuscular injection ofKetamine (20 mg/kg) (Fort Dodge Animal Health, Overland Park, Kans.,USA), together with Xylazine (0.5 mg/kg) (Bayer, Shawnee Mission, Kans.,USA). All rabbits received a urine catheter to prevent any disruptionsfrom signal in the bladder.

Rabbits were placed in a body matrix coil and received isofluraneanesthesia at 1.5% by inhalation and were oxygenated for the remainingof the PET/MR imaging experiment, while vital parameters were monitored.Shortly after injection, images were acquired in a dynamic fashion forthe duration of 1 hour using a clinical 3 Tesla PET/MRI Biograph mMR(Siemens, Munchen, Germany). After scout scans, the PET scan wasinitiated and co-acquired with a radial VIBE MR sequence with thefollowing imaging parameters: TR, 20 ms; TE, 1.89 ms; flip angle, 10degrees; slice thickness, 1.1 mm³. Attenuation correction of PET imageswas done using the built-in MR-based attenuation correction (MR-AC) mapand images reconstructed using the OP-OSEM algorithm. In addition, atime-of-flight (TOF) non-contrast enhanced angiography was performed forlocalization of arterial anatomical landmarks (renal arteries and iliacbifurcation). Imaging parameters were: TR, 1600 ms; TE, 118 ms; flipangle, 140 degrees; ETL, 83; slice thickness, 0.6 mm.

A dynamic contrast-enhanced MRI (DCE-MRI) scan was performed. Blackblood was obtained using a double inversion recovery (DIR) technique. A3D turbo field echo (TFE) sequence with motion sensitized drivenequilibrium (MSDE) preparation for black blood imaging was used toquantify the uptake of a FDA approved gadolinium based CA; gadopentetatedimeglumine (Magnevist, Bayer Healthcare) from the right renal artery tothe iliac bifurcation. Imaging parameters were: TR, 6.2 ms; TE, 2.8 ms;flip angle, 20 degrees; ETL, 80; spatial resolution, 0.6 mm³; FOV, 160mm²; 20 slices; orientation, sagittal. This sequence was used before and10 minutes after CA injection to quantify the CA accumulation in thevessel wall and thus to measure the permeability of the vessel wall.Before and during CA injection, the same sequence was used with 3 signalaverages (time resolution 32s) to perform 3D DCE-MRI, and quantify therate of uptake of CA in the vessel wall. The next day, 24±1.5 hoursafter injection, all rabbits received a 20 minutes static PET-scan,again using a TOF and MR-AC.

Pharmacokinetics and Biodistribution ⁸⁹Zr-LA25 or ⁸⁹Zr-LA24 in Rabbits.

Radioactivity half-lives were determined by drawing blood from the eararteries at 1 and 30 minutes, and at 1, 2, 4, 20, 24 and at sacrificeafter 28 hours. All rabbits were sacrificed by an i.v. injected overdoseof 100 mg/kg sodium pentobarbital and subsequently perfused with 500 mlsaline. After sacrifice, all animals were perfused to make sure no bloodor blood clots remained in the aorta and other organs before excision.Aortas were excised and divided in thoracic, form the aortic root untilthe diaphragm and the abdominal aorta, infra-diaphragmatic until iliacbifurcation, the latter with celiac trunk and renal arteries attached,serving as landmarks. The following organs were harvested: heart, lungs,liver, spleen, kidneys, one adrenal gland, muscle and bone-marrow andwere weighted. All tissues were weighted before counting with a Wizard22480 automatic gamma counter. Radioactivity concentration in tissues wascalculated as percentage of injected dose per gram (% ID/g).

Near-Infrared Fluorescence Imaging.

Twenty-four hours before sacrifice, all rabbits received fluorescentlylabeled high-density lipoprotein (Cy5.5-HDL, ˜1 mg dye per rabbit) in 5ml PBS solution via the marginal ear vein. After sacrifice all aortas,thoracic and abdominal, were placed on thick black paper and imaged witha Xenogen IVIS-200 optical imaging system (Perkin Elmer, Waltham,Mass.). Fluorescence images were acquired with excitation and emissionwavelengths of 680 and 720 nm and a field of view (FOV) of 6.5 cm and22.8 cm using different exposure times.

Autoradiography.

Aortas were placed in a film cassette against a phosphorimaging plate(BASMS-2325, Fujifilm, Valhalla, N.Y.) for 48 h (mouse aortas, rabbitorgans) or 72 h (rabbit aortas) at −20° C. to determine radiotracerdistribution. Luminal autoradiography was performed for 96 h, after oneof the harvested aortas was cut into 20 μm thick sections.Phosphorimaging plates were read at a pixel resolution of 25 μm with aTyphoon 7000IP plate reader (GE Healthcare, Pittsburgh, Pa.).

Image Analysis.

Image analysis for PET-imaging was performed after all data wereprocessed and divided in different time frames using a custom-madeprogram written in Matlab (Mathworks, Natick, Mass.). All data wassubsequently processed using OsiriX Imaging Software (OsiriX Foundation,Geneva, Switzerland) by drawing regions of interest (ROIs) on theinfrarenal abdominal aorta, and major organs (liver, spleen andkidneys). By averaging all acquired ROIs per organ (≥10 per organ), meanSUV_(max) values in each tissue were obtained. All images acquired withDCE-MRI were reformatted in the axial plane for tracing. The vessel walltracing was made on the average image of the dynamic series of DCE-MRIusing Osirix software (OsiriX Foundation, Geneva, Switzerland). Bydrawing an inner and an outer vessel wall contour and computing thedifference between these two, the vessel wall area or Region of Interest(ROI) was measured.

The area under the normalized signal intensity curve (IAUC) wascalculated after two minutes, serving as a time point for data analysis,with a custom-made program written in Matlab (Mathworks, Natick, Mass.).IAUC is a measure of contrast agent extravasation and uptake in the(atherosclerotic) vessel wall. IAUC was calculated on a pixel-by-pixelbasis. MR signal intensity over time was normalized to vertebral musclesignal intensity before the injection of contrast agent. NIRF analysiswas done using Living Image Software (Perkin Elmer, Waltham, Mass.). Allthoracic and abdominal aortas were divided in 10 equal regions ofinterest and quantified as Total Radiant Efficiency in [p/s]/[μW/cm²].

Histological Analysis and Immunostaining of Rabbit Aortic Sections.

Sections of 0.5 cm from the excised abdominal aorta were placed inoptimal cutting temperature (OCT) compound and were cut into 7 μm thicksections that were adjacent to the luminal autoradiography cut section.Sections were stained for hematoxylin and eosin, RAM-11 for macrophagesor Oil red 0 for lipids (Dako, Santa Clara, Calif.). Detailed imageswere made with a Nikon Eclipse E400 microscope, a Nikon DS-U1 camerabox, and a Nikon DS-5 M camera while whole luminal aorta images weremade using an Olympus Stereoscope MVX10.

Statistics.

Statistical analysis was conducted using unpaired t-tests. Linearregression was used for the correlative measures, computing Pearson's rcoefficients to determine the degree of correlation. Data are reportedas mean±standard deviation. P values of <0.05 were consideredstatistically significant. For the calculation of the differentstatistical parameters, Prism (version 6.0, GraphPad Software Inc., LaJolla, Calif.) was used.

Cloning and Characterization of LA25.

LDL was modified with MAA, MDA or CuSO₄ to generate MAA-LDL (FIG. 1A),MDA-LDL or copper-oxidized LDL (Cu-OxLDL) respectively. MAA is aspecific advanced MDA-type adduct that is an immunodominant MDA epitope.To enrich for antibodies, a Fab library was constructed (K/A) fromlymphocytes isolated from umbilical cord blood of 7 newborns andscreened these with MAA-BSA. After 4 rounds of panning with eachlibrary, an enrichment in the newborn library of fully or very neargermline sequences was identified, consistent with minimal non-templatedinsertions in the CDR3 region (FIG. 1B). The resultant Fabs were clonedinto the phagemid pComb3X and LA25 was identified as a leading candidatedue to its specificity for MAA epitopes and optimal expressioncharacteristics. LA24 was identified from the same library, but did notbind any relevant oxidation-specific epitopes present in vivo. LA25 andLA24 plasmids from selected Fab phage clones were codon optimized andtransformed into E. coli C41 (DE3) for production of soluble Fab.

LA25 detected MAA-LDL, but did not detect LDL, Cu-OxLDL or MDA-LDL (FIG.1C). LA24 did not detect any of these epitopes. To assess specificityfor MAA-LDL, competition experiments were performed of LA25 binding toMAA-LDL, which demonstrated specific and near complete ability ofMAA-LDL, but not other competitors, to compete LA25 binding (FIG. 1D).

Immunostaining of Human Advanced and Ruptured Plaques and Debris fromDistal Protection Devices with LA25.

Immunostaining of human advanced fibroatheromas and ruptured coronaryplaques with LA25 showed specificity in the necrotic core (FIG. 20).Interestingly, LA25 also stained the thrombus adjacent to the plaquerupture. Thrombi are known to express MDA/MAA epitopes, likely throughsecretion by activated platelets. Debris from distal protection devicesalso stained strongly with LA25, as well as MDA and OxPL epitopes (FIG.20). MAA epitopes did not co-localize with OxPL epitopes in thesespecimens, but MDA epitopes co-localized partially with both MAA andOxPL epitopes.

Pharmacokinetics, Biodistribution and Plaque Specificity ⁸⁹Zr-LA25 inMice.

Initially, the ⁸⁹Zr-labeled Fabs were tested in Apoe^(−/−) mice (22weeks on high-fat diet) to investigate their in vivo behavior andspecificity for MAA epitopes in atherosclerotic plaques. Mice wereintravenously injected with either ⁸⁹Zr-LA25 or ⁸⁹Zr-LA24, which wasused as chemical control. The blood radioactivity half-lives were 29 and13 minutes for LA25 and LA24 respectively, and blood radioactivityconcentrations differed significantly starting from 30 minutes afterinjection (FIG. 22A).

Radioactivity distribution in selected tissues was determined by gammacounting at 4 hours post injection (p.i.). Aortic uptake wassignificantly higher in mice injected with ⁸⁹Zr-LA25 compared with⁸⁹Zr-LA24, 1.56±0.35 vs. 0.41±0.13% ID/g, P<0.0001 (FIG. 22B).Representative autoradiographs of mouse aortas showed homogenousradioactivity distribution in those injected with ⁸⁹Zr-LA24 comparedwith ⁸⁹Zr-LA25, which showed a heterogeneous pattern of uptake with moreintense depositions at the level of typical lesion sites such as theaortic root and the abdominal aorta (indicated by arrows, FIG. 22C).

A high kidney accumulation was found in all mice, indicative of renalclearance (FIG. 22D). Despite the higher renal uptake for ⁸⁹Zr-LA24, thedifference between the two groups was not statistically significant,85.5±20.0 vs. 64.2±16.3, P=0.06. Furthermore, uptake in liver and spleenwas significantly lower in mice injected with ⁸⁹Zr-LA24 compared to⁸⁹Zr-LA25, 0.53±0.12 vs. 3.50±1.67% injected dose/gram tissue (% ID/g),P=0.0006, and 0.63±0.16 vs. 2.69±1.03% ID/g, P=0.0003, respectively.

Dynamic PET/MR Imaging with Ex Vivo Confirmation.

Atherosclerotic rabbits were dynamically scanned for one hourimmediately after intravenous injection of either ⁸⁹Zr-LA25 or ⁸⁹Zr-LA24(FIG. 23A top and bottom, respectively). Standardized uptake values(SUV) were measured in kidney, liver and spleen, at 0-60 minutes and 24hours after injection, showing an increase in kidney uptake and a slightdecrease in liver and spleen uptake for both Fabs (FIG. 23B). Liveruptake was significantly higher over the course of the first hour and at24 hours after injection in rabbits injected with ⁸⁹Zr-LA25 compared to⁸⁹Zr-LA24. Splenic uptake was also significantly higher at 30 and 40minutes and 24 hours after injection in rabbits injected with ⁸⁹Zr-LA25.Kidney uptake, on the other hand, was significantly higher for ⁸⁹Zr-LA24during the first hour and 24 hours post injection. Indeed, bloodradioactivity half-life was longer for ⁸⁹Zr-LA25, 2.2 h vs. 1.1 h for⁸⁹Zr-LA24 (FIG. 23C). Thus, as was seen in mice (FIG. 22A), the bloodtime-activity curve for ⁸⁹Zr-LA25 in rabbits also showed delayedclearance. Importantly, PET/MR quantification results were corroboratedby ex vivo gamma counting, with a significantly higher kidney uptake for⁸⁹Zr-LA24 compared to ⁸⁹Zr-LA25.

All rabbits were sacrificed 28 hours post injection and tissuesharvested after thorough perfusion. Radioactivity counting revealed asignificantly higher aortic uptake for ⁸⁹Zr-LA25 compared to ⁸⁹Zr-LA24in rabbits with atherosclerosis, 0.022±0.003 vs. 0.006±0.001% ID/g,P<0.0001 (FIG. 23D). Autoradiography corroborated earlier results inmice, and a heterogeneous radioactivity distribution pattern was foundin the ⁸⁹Zr-LA25 group in contrast with a homogenous distribution foundfor ⁸⁹Zr-LA24 (FIG. 23E).

Phenotyping of Atherosclerotic Plaques in Rabbits.

To non-invasively assess disease burden in healthy NZW rabbits versusNZW rabbits with atherosclerosis, the novel tracer ⁸⁹Zr-LA25 wascombined with previously validated non-invasive imaging protocols forclinical PET/MR imaging. In addition, different hallmarks of advancedatherosclerotic plaques, i.e. oxidation specific epitopes, plaque area,inflammation and neovascularization, and assessed inflammation in anearlier session by FDG-PET were simultaneously measured.

Uptake of ⁸⁹Zr-LA25 was evaluated in vivo by an additional PET/MR staticscan 24 hours after injection, when blood signal was low based onpharmacokinetic data.

Representative aortic coronal fused PET/MR images are shown in FIG. 24A(left), in control and atherosclerotic rabbits injected with ⁸⁹Zr-LA25.Uptake in the vessel wall was quantified by drawing regions of interest(ROI) on the aorta from the left renal artery to the iliac bifurcation.Radioactivity uptake was significantly higher for ⁸⁹Zr-LA25 in rabbitswith atherosclerosis compared with healthy controls, 0.33±0.1 vs.0.25±0.08 g/ml, P=0.0003 (FIG. 24A, right). The PET/MR findings wereconfirmed by autoradiography, showing a heterogeneous deposition patternin atherosclerotic aortas compared with the homogenous lower uptakefound in control aortas (FIG. 24B, left) and ex vivo gamma counting (28hours post injection) revealed a significantly higher uptake for⁸⁹Zr-LA25 in atherosclerotic aortas (0.022±0.003 vs. 0.005±0.001% ID/g,P<0.0001, FIG. 24B right). Moreover, correlations between thetarget-to-blood ratio (TBR) of aortas as determined by PET (as ratio towithdrawn blood) and gamma counting showed a significant positivecorrelation (r=0.92, P<0.0001).

A significantly larger plaque area in the abdominal aorta was measuredfor rabbits with atherosclerosis compared to their healthy controlcounterparts (0.29±0.06 vs. 0.11±0.03 mm², P=0.0014, FIG. 24C).Forty-eight hours prior to ⁸⁹Zr-LA25 injection, inflammation wasassessed by quantifying ¹⁸F-FDG uptake in the abdominal aorta by drawingROIs on the same aortic regions as performed for ⁸⁹Zr-LA25-PET. Uptakewas significantly higher in atherosclerotic rabbits compared to healthycontrols (1.95±0.19 vs. 0.36±0.02 g/ml, P<0.0001, FIG. 24D). Dynamiccontrast enhanced MRI (DCE-MRI) was used to evaluate vascularpermeability, by quantifying the uptake of Magnevist in the abdominalaorta, which was significantly higher in atherosclerotic rabbitscompared to controls (4.28±1.52 vs. 1.81±1.42, P<0.0001), measured asthe intensity area under the curve (IAUC) 2 minutes after injection(FIG. 24E). A fluorescently labeled reconstituted high-densitylipoprotein (rHDL) nanoparticle was used as a macrophage mapping agentthat was injected 24 hours before sacrifice. After euthanasia, allaortas were excised after thorough perfusion to prevent blood clots andimaged by near infrared fluorescence (NIRF). Fluorescence intensityrevealed an approximately 100-fold significant increase inatherosclerotic aortas compared to controls (95±36×10⁹ vs. 1.14±0.31×10⁹μW/cm, P<0.0001, FIG. 24F). In addition, NIRF imaging showed aheterogeneous fluorescence signal distribution in aortas from diseasedrabbits, indicative of accumulation in atherosclerotic lesions.

Of note, a strong correlation between ⁸⁹Zr-LA25 radioactivity and rHDLNIRF intensity in aortas (r=0.94, P=0.0005) was observed, possiblysuggesting a certain degree of macrophage uptake of ⁸⁹Zr-LA25. Inaddition, uptake of ⁸⁹Zr-LA24 by gamma counting and rHDL NIRF intensityshowed a significant negative correlation (r=−0.83 P=0.01). Furthermore,FDG uptake has been correlated with macrophage burden in plaques. Inline with this, strong correlations was found between rHDL fluorescenceintensity and PET-derived FDG uptake (r=0.85, P<0.0001), and ex vivoquantified ⁸⁹Zr-LA25 uptake and PET-derived FDG uptake (r=0.97,P<0.0001). Moreover, a significant correlation between ⁸⁹Zr-LA25radioactivity and plaque area, as determined by T2-weighted MRI, wasfound (r=0.91, P=0.02). However, uptake of ⁸⁹Zr-LA25 in the aorta showedno correlation with permeability as determined by DCE-MRI (IAUC),Pearson r=0.62, P=0.1.

Ex Vivo Plaque Characterization.

After rabbits were sacrificed for ex vivo validation, one abdominalaorta was divided into several different pieces and processed forhistology. The first section in a set of slides was used for luminalautoradiography and adjacent sections were stained for vessel wall areawith conventional hematoxylin & eosin (H&E), RAM-11 for macrophages andOil red 0 for lipid content; representative images are shown in FIG. 25.After the integrated density (mean grey value) area of ⁸⁹Zr-LA25 wasdetermined for all luminal autoradiography, correlations on the adjacentsections were calculated to be r=0.93 (P<0.0001) for vessel wall area,r=0.74 (P=0.0004) for macrophages and r=0.70 (P=0.0008) for lipids (FIG.25).

A number of embodiments have been described herein. Nevertheless, itwill be understood that various modifications may be made withoutdeparting from the spirit and scope of this disclosure. Accordingly,other embodiments are within the scope of the following claims.

1. An isolated antibody or antibody fragment that recognizes and bindsto an MAA oxidized specific epitope (OSE)(MAA-OSE), wherein the antibodyor antibody fragment comprises a variable heavy chain (V_(H)) domainand/or a variable light chain (V_(L)) domain, and wherein, (a) the V_(H)domain comprises a sequence selected from the group consisting of: (i)at least one CDR selected from the group consisting of SEQ ID NO:25, 26and 27; (ii) at least one CDR selected from the group consisting of 28,29 and 30; (iii) at least one CDR selected from the group consisting ofSEQ ID NO:31, 32 and 33; (iv) at least one CDR selected from the groupconsisting of SEQ ID NO:34, 35 and 36; and (v) at least one CDR selectedfrom the group consisting of SEQ ID NO:37, 38 and 39; and/or (b) theV_(L) domain comprises a sequence selected from the group consisting of:(i) at least one CDR selected from the group consisting of SEQ ID NO:40,41 and 42; (ii) at least one CDR selected from the group consisting ofSEQ ID NO:43, 44 and 45; (iii) at least one CDR selected from the groupconsisting of SEQ ID NO:46, 47 and 48; (iv) at least one CDR selectedfrom the group consisting of SEQ ID NO:49, 50 and 52; and (v) at leastone CDR selected from the group consisting of SEQ ID NO:52, 53 and 54.2. The antibody of claim 1, wherein the antibody comprises a variableheavy chain sequence of (a) selected from the group consisting of: (1)SEQ ID NO:4; (2) SEQ ID NO:8; (3) SEQ ID NO:12; (4) SEQ ID NO:16; and(5) SEQ ID NO:20.
 3. The antibody of claim 1 or 2, wherein the antibodycomprises a variable light chain sequence of (b) selected from the groupconsisting of: (6) SEQ ID NO:2; (7) SEQ ID NO:6; (8) SEQ ID NO:10, (9)SEQ ID NO:14; and (10) SEQ ID NO:18.
 4. The antibody of claim 1, whereinthe antibody comprise a variable light chain sequence that is at least98% identical to SEQ ID NO:2 and a variable heavy chain sequence that isat least 98% identical to SEQ ID NO:4.
 5. The antibody of claim 1,wherein the antibody comprise a variable light chain sequence that is atleast 98% identical to SEQ ID NO:6 and a variable heavy chain sequencethat is at least 98% identical to SEQ ID NO:8.
 6. The antibody of claim1, wherein the antibody comprise a variable light chain sequence that isat least 98% identical to SEQ ID NO:14 and a variable heavy chain thatis at least 98% identical to SEQ ID NO:16.
 7. The antibody of claim 1,wherein the antibody comprise a variable light chain that is at least98% identical to SEQ ID NO:18 and a variable heavy chain that is atleast 98% identical to SEQ ID NO:20.
 8. The antibody of claim 1, whereinthe antibody is non-human and comprises a variable light chain that isat least 98% identical to SEQ ID NO:10 and a variable heavy chain thatis at least 98% identical to SEQ ID NO:12.
 9. The antibody of claim 8,wherein the antibody is humanized.
 10. (canceled)
 11. A single chainvariable fragment (“scFv”) that recognizes an MAA-OSE and comprises (A)a V_(H) domain containing a sequence selected from the group consistingof: (i) at least one CDR selected from the group consisting of SEQ IDNO:25, 26 and 27; (ii) at least one CDR selected from the groupconsisting of 28, 29 and 30; (iii) at least one CDR selected from thegroup consisting of SEQ ID NO:31, 32 and 33; (iv) at least one CDRselected from the group consisting of SEQ ID NO:34, 35 and 36; and (v)at least one CDR selected from the group consisting of SEQ ID NO:37, 38and 39; and (B) a V_(L) domain containing a sequence selected from thegroup consisting of: (i) at least one CDR selected from the groupconsisting of SEQ ID NO:40, 41 and 42; (ii) at least one CDR selectedfrom the group consisting of SEQ ID NO:43, 44 and 45; (iii) at least oneCDR selected from the group consisting of SEQ ID NO:46, 47 and 48; (iv)at least one CDR selected from the group consisting of SEQ ID NO:49, 50and 52; and (v) at least one CDR selected from the group consisting ofSEQ ID NO:52, 53 and
 54. 12. The antibody fragment of claim 11, whereinthe scFv is soluble under physiological conditions.
 13. An antibodycomprising a variable light chain and variable heavy chain sequenceselected from the group consisting of: (a) SEQ ID NO:2 and 4; (b) SEQ IDNO:6 and 8; (c) SEQ ID NO:10 and 12; (d) SEQ ID NO:14 and 16; and (e)SEQ ID NO:18 and
 20. 14. The antibody of claim 13, wherein the variablelight chain comprises SEQ ID NO:10 and the variable heavy chain compriseSEQ ID No:12 and an Fc region is human or humanized.
 15. (canceled) 16.A polynucleotide that encodes an antibody, or antibody fragment ofclaim
 1. 17. A vector comprising a polynucleotide sequence selected fromthe group consisting of: (a) SEQ ID NO:1 and/or 3; (b) SEQ ID NO:5and/or 7; (c) SEQ ID NO:9 and/or 11; (d) SEQ ID NO:13 and/or 15; and (e)SEQ ID NO:17 and/or
 19. 18. A host cell transformed with thepolynucleotide of claim
 16. 19. A host cell transformed with a vector ofclaim
 17. 20. A transgenic animal, comprising a polynucleotide of claim16.
 21. A method of treating a subject with an MAA-related disease ordisorder comprising administering an antibody or antibody fragment ofclaim 1 to the subject, wherein the antibody or antibody fragment bindsto and inhibits the biological effect caused by an MAA adduct.
 22. Themethod of claim 21, wherein the antibody or antibody fragment comprisesa sequence selected from the group consisting of: (a) SEQ ID NO:2 and 4;(b) SEQ ID NO:6 and 8; (c) SEQ ID NO:14 and 16; and (d) SEQ ID NO:18 and20.
 23. The method of claim 22, wherein the subject is a human.
 24. Themethod of claim 21, wherein the subject has a cardiovascular disease ordisorder, a fatty liver disease or disorder, an acute lung injurydisease or disorder, or rheumatoid arthritis disease or disorder.
 25. Amethod of treating oxidative stress in a subject comprisingadministering an antibody or antibody fragment of claim 1 or 13 to thesubject, wherein the antibody binds to and inhibits the biologicaleffect caused by an MAA adduct.
 26. A method of diagnosing aninflammatory fatty liver disease or disorder comprising contacting asubject with an antibody or antibody fragment of claim 1, wherein theantibody is labeled with a detectable label and imaging the subject todetermine the amount or presence of the antibody in the liver of thesubject.
 27. The method of claim 26, wherein if the amount of theantibody in the liver exceeds a normal control amount then the subjecthas an inflammatory liver disease.
 28. The method of claim 26, whereinthe inflammatory liver disease is non-alcoholic fatty liver disease. 29.The method of claim 26, wherein the inflammatory liver disease isnon-alcoholic steatohepatitis (NASH).
 30. A method of diagnosing aMAA-related disease or disorder in a subject comprising obtaining asample from the subject and contacting the sample with an antibody orantibody fragment of claim 1 and measuring the amount of antibody orantibody fragments bound to and MAA adduct in the sample compared to anormal control sample, wherein an amount of antibody or antibodyfragment bound to an MAA adduct in the sample is greater than thecontrol, the subject has an MAA-related disease or disorder.
 31. Amethod of treating or inhibiting atherogenesis in a subject, the methodcomprising administering to the subject an antibody or antibody fragmentof claim 1, wherein the antibody binds to an inhibits that uptake and/orinflammatory response caused by an MAA adduct.
 32. The method of claim31, wherein the MAA adduct is associated with a molecule in anatherosclerotic plaque.
 33. A method for treating atherosclerosis and/orfatty liver disease in a subject, the method comprising administering tothe subject antibodies or antibody fragments of claim 1 thatspecifically bind an MAA adduct, wherein the antibodies or antibodyfragments are in a pharmaceutically acceptable carrier.
 34. A method ofinhibiting the progression of non-alcoholic fatty liver disease tonon-alcoholic steatoheptatis (NASH) comprising administering to asubject in need of such treatment and antibody or antibody fragment ofclaim 1 in an amount to reduce inflammation and to improve biomarkers ofliver function.